Combined screening for autoantibodies to IA-2 and antibodies to glutamic acid decarboxylase in first degree relatives of patients with IDDM. The DENIS Study Group. Deutsche Nikotinamid Interventions-Studie

To determine the value of antibodies to the intracytoplasmic domain of the tyrosine phosphatase IA-2 (anti-IA-2ic) and glutamic acid decarboxylase (GADA) for identification of subjects at risk for insulin-dependent diabetes mellitus (IDDM) we investigated 1238 first degree relatives of patients with IDDM for the presence of anti-IA-2ic and GADA and compared the results with cytoplasmic islet cell antibodies (ICA). Anti-IA-2ic were observed in 54 (4.4%) first degree relatives, in 51 of 86 (59.3%) ICA positive relatives and in 3 of 4 individuals who developed overt IDDM within a follow-up period of 1 to 28 months. GADA were found in 78 of 1238 (6.3%) first degree relatives. They were detected in 22 of 35 (62.9%) sera with ICA alone and in 1 of 3 subjects with anti-IA-2ic in the absence of ICA. Of the 1238 subjects 37 (3.0%) sera were positive for all three antibodies. Both anti-IA-2ic and GADA were positively correlated with high levels of ICA. Anti-IA-2ic and GADA were detected in 39.1 and 47.8% of subjects with ICA of less than 20 Juvenile Diabetes Foundation units (JDF-U) but in 66.7 and 76.2% of individuals with ICA of 20 JDF-U or more, respectively (p < 0.05). The levels of ICA and GADA in first degree relatives with at least one additional marker were significantly higher than in subjects with ICA alone (p < 0.005) or GADA alone (p < 0.03). The combination of anti-IA-2ic and GADA identified 84.9% of all ICA positive subjects and 93.7% of individuals with high level ICA (> or = 20 IDF-U). All 4 individuals who progressed to IDDM had either IA-2ic or GADA. Our data indicate that primary screening for anti-IA-2ic and GADA provides a powerful approach with which to identify subjects at risk for IDDM in large-scale population studies which may represent the basis for the design of new intervention strategies.     

Islet cell autoimmunity in white and black children and adolescents with IDDM

OBJECTIVE: To compare the frequency of islet cell antibodies (ICA) and antibodies to GAD65 and IA-2(ICA512) between black and white children and adolescents at the diagnosis of IDDM in a large consecutive series of cases from Children's Hospital of Pittsburgh. RESEARCH DESIGN AND METHODS: ICA and antibodies to GAD65 and IA-2 were measured in 437 white and black children and adolescents who were diagnosed with IDDM at < 19 years of age at Children's Hospital of Pittsburgh from January 1983 to December 1985, from January to December 1989, and from January 1996 to December 1997. RESULTS: The prevalence of ICA(H), GAD65, and IA-2 antibodies was significantly lower in blacks than whites at onset of the disease. In contrast, the prevalence of ICA(R) alone was higher in blacks. None of the antibodies were present in 12% of the blacks compared with 4% in whites. The same pattern was seen in both sexes. The prevalence of antibodies in white patients with onset of IDDM at <11 years of age was no different than in those who developed IDDM during adolescence. In contrast, black patients showed a significantly lower prevalence of almost all antibodies in the adolescent group. CONCLUSIONS: Black adolescents were more likely to not have antibodies, suggesting either that they have a nonautoimmune type of diabetes or that antibodies are not being detected by these assays.     

Coeliac disease: frequent occurrence after clinical onset of insulin-dependent diabetes mellitus. Childhood Diabetes in Finland Study Group

Coeliac disease was searched for in a series of 776 children with newly diagnosed IDDM. During the follow-up of 2 to 3 years from diagnosis, reticulin and gliadin antibodies were measured, and a jejunal biopsy was performed in those cases with high levels of antibodies; 19 children were identified with coeliac disease, giving the prevalence of 2.4%. In only one case had coeliac disease been diagnosed before IDDM. Nine patients with proven coeliac disease were negative for antibodies when IDDM was diagnosed, but became positive within 24 months. All patients found to have coeliac disease were positive for IgA reticulin antibodies, but only 12 of 18 (67%) showed a high level of IgA gliadin antibodies. Of the 18 patients genotyped for HLA DR locus, 14 (78%) were positive for DR3 and 10 (56%) were positive for DR4. DQB1*0201 allele was present in 17 of 18 patients (94%). Coeliac disease in children with IDDM tends to develop soon after diabetes is diagnosed. Routine screening for coeliac disease is recommended repeatedly during the first years after the diagnosis of IDDM.     

Islet cell antibodies are less predictive of IDDM among unaffected children in the general population than in sibs of children with diabetes. The Childhood Diabetes in Finland Study Group

OBJECTIVE: To examine the controversial issue of whether islet cell antibodies (ICAs) have a higher predictive value for progression to clinical IDDM in first-degree relatives of patients with diabetes than in the general population. RESEARCH DESIGN AND METHODS: ICAs were analyzed with standard immunofluorescence in two population-based groups: 765 sibs of children with recent-onset diabetes and 1,212 unaffected Finnish children <20 years of age at initial screening. Those positive for ICAs were additionally tested for antibodies to GAD (GADAs) and the protein tyrosine phosphatase-related IA-2 antigen (IA-2As). Subsequently, these subjects were observed for the manifestation of clinical IDDM over the next 7 years. RESULTS: The frequency of both detectable ICAs and ICA levels > or =20 Juvenile Diabetes Foundation units (JDF U) was significantly higher among the sibs than in the general population (7.8 vs. 4.1% and 4.8 vs. 2.0%, respectively; P < 0.001). The prevalence of GADAs (37/60 vs. 3/48; P < 0.001) and IA-2As (31/60 vs. 0/48; P < 0.001) was increased among ICA-positive sibs compared with ICA-positive individuals from the background population. Over the next 7 years, 24 sibs (3.1%) and 3 unrelated children positive for ICAs (0.3%) progressed to clinical diabetes. The positive predictive value of ICAs was thus 6% in the general population and 40% among the sibs (P < 0.001), or 13 and 59%, respectively (P < 0.001), with an antibody cutoff level of 20 JDF U. The positive predictive value was related to the number of positive autoantibodies in sibs, which was 57% in those with three antibodies, 50% in those with two antibodies, and only 6% in those with ICAs alone. CONCLUSIONS: These data show that the frequency of multiple autoantibodies is substantially lower in ICA-positive children representing the general population than in ICA-positive sibs of children with IDDM. As a consequence, the predictive value of ICAs for IDDM is higher in sibs of affected children than in the general population. This finding must be taken into account when planning intervention trials aimed at preventing or delaying the manifestation of clinical diabetes in individuals from the general population who test positive for ICAs.     

UKPDS 25: autoantibodies to islet-cell cytoplasm and glutamic acid decarboxylase for prediction of insulin requirement in type 2 diabetes. UK Prospective Diabetes Study Group

BACKGROUND: Autoantibodies to islet-cell cytoplasm (ICA) and glutamic acid decarboxylase (GADA) can occur in apparently typical, non-insulin dependent diabetes mellitus (type 2). We investigated whether the presence of either or both antibodies characterises a subtype of diabetes and provides better prediction of requirement for insulin therapy by 6 years' follow-up than clinical variables. METHODS: We measured ICA and GADA at diagnosis of diabetes in a representative population of 3672 white patients with type 2 diabetes, aged between 25 and 65 years. The phenotype was assessed by age of onset, body-mass index, percentage haemoglobin A1c (HbA1c), and islet beta-cell function. We investigated the need for insulin therapy among 1538 patients not assigned insulin and followed up for 6 years from diagnosis. FINDINGS: The proportion of patients with ICA and GADA decreased with increasing age at diagnosis (from 33 [21%] of 157 patients aged 25-34 [corrected] to 66 [4%] of 1769 aged 55-65 for ICA; from 53 [34%] to 122 [7%] for GADA). Among patients younger than 35 at diagnosis, those with ICA or GADA had lower body-mass index than those without (mean 24.9 [SD 6.0] vs 31.7 [7.3] kg/m2; p < 0.0001 and had higher percentage of HbA1c (9.7 vs 8.7%, p < 0.05). 94% of patients with ICA and 84% of those with GADA required insulin therapy by 6 years, compared with 14% of those without the antibodies (p < 0.0001). Among patients older than 55 at diagnosis, the difference between those with and without antibodies in body-mass index was smaller (27.2 [5.4] vs 28.6 [4.8] kg/m2, p < 0.001); 44% of those with ICA, 34% of those with GADA, and 5% with neither antibody required insulin therapy by 6 years (p < 0.0001). Among patients older than 45 years, body-mass index and HbA1c provided little predictive information for insulin requirement, whereas the positive predictive values of GADA (> or = 60 U/L) alone, or both GADA (> or = 20 U/L) and ICA (> 5 U/L), for insulin therapy were 52% and 68%. INTERPRETATION: Among young adults with type 2 diabetes, the phenotype of those with ICA or GADA antibodies was similar to that of classic juvenile-onset insulin-dependent diabetes, and either phenotype or antibodies predicted insulin requirement. In older adults, the phenotype was closer to that of patients without antibodies and only the presence of antibodies predicted an increased likelihood of insulin requirement.     

Associations of GAD65- and IA-2- autoantibodies with genetic risk markers in new-onset IDDM patients and their siblings. The Belgian Diabetes Registry

OBJECTIVE: To investigate the association of GAD (65-kDa) autoantibodies (GAD65-Abs) and IA-2 autoantibodies (IA-2-Abs) with human leukocyte antigen (HLA)-DQ and insulin gene (INS) risk markers in patients with recent-onset IDDM and their siblings. RESEARCH DESIGN AND METHODS: Blood was sampled from 608 recent-onset IDDM patients and 480 siblings, aged 0-39 years and consecutively recruited by the Belgian Diabetes Registry, to determine GAD65- and IA-2-Ab (radiobinding assay), HLA-DQ- (allele-specific oligonucleotyping), and INS-genotypes (restriction fragment length polymorphism analysis; siblings, n = 439). RESULTS: At the onset of IDDM, GAD65-Abs were preferentially associated with two populations at genetic risk but only in the 20- to 39-year age-group: 1) their prevalence was higher in carriers of DQA1*0301-DQB1*0302 (88 vs. 73% in non[DQA1*0301-DQB1*0302], P = 0.001), and 2) an association was found in patients lacking this haplotype but carrying DQA1*0501-DQB1*0201, together with INS I/I (87 vs. 54% vs. non[INS I/I], P = 0.003). Siblings of IDDM patients also presented the association of GAD65-Abs with DQA1*0301-DQB1*0302 (13 vs. 2% non[DQA1*0301-DQB1*0302], P < 0.001), while associations with the second genetic risk group could not yet be assessed. At the onset of IDDM, IA-2-Ab prevalence was higher in carriers of DQA1*0301-DQB1*0302 (69 vs. 39% non[DQA1*0301-DQB1*0302], P < 0.001) but not of DQA1*0501-DQB1*0201 or INS I/I. This association was present in both the 0- to 19- and the 20- to 39-year age-groups. It was also found in siblings of IDDM patients (4 vs. 0% non[DQA1*0301-DQB1*0302], P < 0.001). CONCLUSIONS: Both GAD65- and IA-2-Abs exhibit higher prevalences in presence of HLA-DQ- and/or INS-genetic risk markers. Their respective associations differ with age at clinical onset, suggesting a possible usefulness in the identification of subgroups in this heterogeneous disease.     

Persistent GAD 65 antibodies in longstanding IDDM are not associated with residual beta-cell function, neuropathy or HLA-DR status

Persistent humoral autoimmunity to the enzyme glutamic acid decarboxylase (GAD) has been described in a substantial proportion of patients with insulin-dependent diabetes mellitus (IDDM) of long duration. The source of the stimulus for this autoimmune reactivity is still unknown. Because the GAD 65 isoform is mainly expressed in pancreatic beta-cells and in the nervous system we investigated in the present study of the largest number of well characterized patients with longstanding IDDM (n = 105; median duration: 21 years; range: 10-46 years) the presence of autoantibodies to GAD 65 and their relationship to a residual C-peptide response or peripheral and autonomic neuropathy. Additionally we studied the HLA-DR status relative to GAD 65 antibodies in 86 out of the 105 individuals. One hundred healthy control subjects and 100 recent onset IDDM patients were also studied for GAD 65 antibodies. GAD 65 antibodies were detected in a radioligand-binding-assay with recombinant human GAD 65 and were present in 32% of the long-term diabetic patients, 82% of the recent onset IDDM patients and in 3% of the healthy control subjects. A preserved C-peptide response to i.v. glucagon (Hendriksen criteria) was observed in 23% of the long-term IDDM patients. Autonomic neuropathy and peripheral neuropathy was identified using criteria based on both symptoms and formal testing giving a frequency of 67% vs 79%. The HLA specific DR 4/X was observed in 47% and HLA-DR 3/X in 22% of the long-term IDDM patients. Patients who were heterozygous for DR3/DR4 were found in 23% of the cases. GAD 65 antibodies were significantly less frequent in the long-term IDDM patients compared to recent onset IDDM (p < 0.001), and diabetes duration showed a significant negative correlation with GAD 65 antibody index levels (r = 0.22, p < 0.01). Interestingly, GAD 65 antibodies were not significantly correlated either with residual beta-cell function or neuropathy and no particular HLA-DR status was associated with persistent GAD 65 antibodies. In conclusion neither residual beta-cell function nor diabetic neuropathy or a certain HLA-DR specificity are exclusively associated with persistent autoimmunity directed to GAD 65 in longstanding IDDM. The stimulus for the persistent humoral immune response and its significance for the disease process and its complications remain to be established.     

Combined use of autoantibodies (IA-2 autoantibody, GAD autoantibody, insulin autoantibody, cytoplasmic islet cell antibodies) in type 1 diabetes: Combinatorial Islet Autoantibody Workshop

The aim of this workshop was to assess the ability of individual autoantibody (ab) assays and their use in combination to discriminate between type 1 diabetic and control sera. Coded aliquots of sera were measured in a total of 119 assays by 49 participating laboratories in 17 countries. The sera were from 51 patients with new onset type 1 diabetes and 101 healthy control subjects with no family history of diabetes. In the final analysis, data on diabetic sera were restricted to 43 subjects younger than age 30 years. The laboratories were asked to report results for these sera using their currently available anti-islet autoantibody assays. In addition, they were asked to combine information from their assays to classify sera as having high, moderate, or low probability of originating from a patient with type 1 diabetes. Actual strategies for combining assays were determined by each laboratory. There were no significant differences in sensitivity among 19 radioimmunoassays (RIAs) for IA-2 autoantibodies (cytoplasmic islet cell antibody [ICA] 512) using different constructs that included the intracellular portion of the molecule (mean sensitivity 73%). However, an enzyme-linked immunosorbent assay (ELISA) using the extracellular portion of the IA-2 molecule did not discriminate between diabetic and control sera. Among GAD autoantibody assays that achieved sensitivity >70%, 26 were RIAs and one was an ELISA. When the sera were ranked according to their autoantibody levels, the concordance for insulin autoantibodies (IAAs) in different laboratories was markedly less than for IA-2ab and GADab. Using a combination of autoantibody assays, several laboratories achieved excellent discrimination between diabetic and control sera (sensitivity up to 80% with false-positive rate of 0%). A variety of strategies for combining information from different assays were successful (e.g., those including and excluding ICA), and no one strategy emerged as clearly superior. In conclusion, IA-2/ICA512 autoantibodies are a marker of type 1 diabetes and can be measured consistently by most assays. Several different strategies for combining assays achieved high sensitivity with a low false-positive rate.     

Oral immunisation as a strategy for enhancing corneal allograft survival

Marked differences have been reported in the prevalence of glutamic acid decarboxylase (GAD) antibodies between Caucasian (63-84%) and Japanese (30-50%) or Asian (5-50%) IDDM patients. Using a new immunoprecipitation assay based on 125I-labelled recombinant human GAD65 we have reassessed prevalence of GAD65 antibodies in Japanese patients. We also assessed prevalence of IA-2 antibodies. GAD65 antibodies were detected in 83.3% of sera taken within 1 year of onset, comparable to the prevalence reported in Caucasian patients. Positivity decreased to 66.7% after 2 to 3 years and to 54.3% after 3 years from onset, still higher than previously reported Asian prevalence. Except in one patient, high antibody levels persisted chronically, up to 12 years. There was no difference in the prevalence of GAD65 antibodies between Japanese IDDM patients with and without autoimmune thyroid disease (AITD). IA-2 antibodies were detected in 64.7% of sera taken within 1 year of onset. Prevalence of IA-2 antibodies was lower than that of GAD65 antibodies. The difference in positivity in Asian IDDM patients between present and previous reports arose from the sensitivity of our assay for GAD65 antibodies. Additionally, the patients we studied had classic IDDM with a well-defined onset. We conclude that prevalence of GAD65 antibodies in Japanese IDDM patients is comparable to that in Western studies. There was no relationship of GAD65 antibody positivity to coexistence of AITD. Our results suggest that autoimmunity is the most significant cause of Japanese IDDM.     

Association between HLA and islet cell antibodies in diabetic patients with a mitochondrial DNA mutation at base pair 3243

Islet cell antibodies (ICA), autoantibodies to glutamic acid decarboxylase (GAD) and HLA genotypes were examined in 31 patients with diabetes and a mitochondrial gene mutation located at base pair 3243 (mtDNA 3243 mutation). ICA was detected in 42% (13/31) of these patients compared to 0 of 90 among healthy control subjects. The ICA showed a "non-restricted" pattern of staining in all 13 ICA-positive patients. In a sensitive radioligand assay only 2 of 31 (6%) diabetic patients with the mutation were positive for both GAD65 autoantibodies and ICA, while the remaining 29 patients were GAD65 antibody negative. The ICA-positive patients had an increased frequency of the HLA-DQA1*0301 allele compared to control subjects (p < 0.05). Of the diabetic patients with the mutation 45% (14/31) had progressive clinical course of beta-cell failure. These results indicate that patients with an mtDNA 3243 mutation may develop islet autoimmunity associated with ICA and GAD autoantibodies. We hypothesize that the presence of HLA-DQA1*0301 in individuals with the mtDNA 3243 mutation increases the risk for diabetes and associated autoantibodies against islet cell antigens.     

Evaluation of ICA512As in combination with other islet cell autoantibodies at the onset of IDDM

OBJECTIVE: The ICA512 pancreatic islet autoantigen is a putative tyrosine phosphatase that is co-identified with the earlier described 40-kDa autoantigen. We report the frequency of autoantibodies to islet cell antigen 512 (ICA512As) in recent-onset IDDM and compare this with other islet cell autoantibodies, including those to GAD (GADAs), insulin (IAAs), and islet cell cytoplasm (ICAs) identified by immunofluorescence. RESEARCH DESIGN AND METHODS: Sera from 232 children aged between 9 months and 14.9 years collected within 14 days of diagnosis were tested for ICA512As by a radioimmunoprecipitation assay. The results were compared with previously reported data for GADAs (n = 232), IAAs (n = 167), and ICAs (n = 230). RESULTS: The frequency of a positive result for ICA512As in children with newly diagnosed IDDM was 60%. The frequency was greater for children with an age of onset between 5 and 10 years (69%) than for children aged < 5 years (49%) and aged between 10 and 15 years (56%). The frequencies for other autoantibody reactivities were 69% for GADAs, 65% for IAAs, and 70% for ICAs. A combination of positive results for ICA512As, GADAs, and IAAs gave a sensitivity for the diagnosis of childhood IDDM of 95%, which was not significantly increased by a positive result for ICAs (96%). CONCLUSIONS: Our results further establish that positivity in a combination of tests is more valuable for the prediction of IDDM than a result for any single autoantibody and that the age of the patient should be considered when selecting the combination of tests to use.     

Cytoplasmic islet cell antibodies recognize distinct islet antigens in IDDM but not in stiff man syndrome

Cytoplasmic islet cell antibodies are well-established predictive markers of IDDM. Although target molecules of ICA have been suggested to be gangliosides, human monoclonal ICA of the immunoglobulin G class (MICA 1-6) produced from a patient with newly diagnosed IDDM recognized glutamate decarboxylase as a target antigen. Here we analyzed the possible heterogeneity of target antigens of ICA by subtracting the GAD-specific ICA staining from total ICA staining of sera. This was achieved 1) by preabsorption of ICA+ sera with recombinant GAD65 and/or GAD67 expressed in a baculovirus system and 2) by ICA analysis of sera on mouse pancreas, as GAD antibodies do not stain mouse islets in the immunofluorescence test. We show that 24 of 25 sera from newly diagnosed patients with IDDM recognize islet antigens besides GAD. In contrast, GAD was the only islet antigen recognized by ICA from 7 sera from patients with stiff man syndrome. Two of these sera, however, recognized antigens besides GAD in Purkinje cells. In patients with IDDM, non-GAD ICA were diverse. One group, found in 64% of the sera, stained human and mouse islets, whereas the other group of non-GAD ICA was human specific. Therefore, mouse islets distinguish two groups of non-GAD ICA and lack additional target epitopes of ICA besides GAD. Longitudinal analysis of 6 sera from nondiabetic ICA+ individuals revealed that mouse-reactive ICA may appear closer to clinical onset of IDDM in some individuals. Mouse-reactive ICAs, however, remained absent in 36% of the patients at diagnosis of IDDM.     

Validity of screening for individuals at risk for type I diabetes by combined analysis of antibodies to recombinant proteins

OBJECTIVE: To determine whether screening for the presence of multiple antibody markers for IDDM is effective at identifying individuals with high risk for disease development. RESEARCH DESIGN AND METHODS: Antibodies to GAD and the tyrosine phosphatase-like protein 1A-2 were determined in sequential serum samples from 44 first-degree relatives of IDDM patients, identified as possessing islet cell antibody (ICA) and/or insulin autoantibody (IAA), who were followed prospectively for IDDM development, ICA, IAA, and antibodies to GAD and 1A-2 were also determined in 93 cases of new-onset nonfamilial IDDM. RESULTS: The presence of two or more antibodies in addition to ICA or IAA conferred high risk (61%) for development of IDDM within 5 years of entry into the study and identified 89% of those who have developed IDDM on current follow-up. None of the relatives positive for ICA or IAA alone, in the absence of other antibody markers, have developed IDDM. Antibodies to islet antigens could both appear and disappear in follow-up samples obtained after entry into the study. The majority (60%) of young (< 16 years), sporadic cases of IDDM had multiple antibodies to islet antigens, but this proportion was lower in older patients (37%). CONCLUSIONS: A screening strategy based on the analysis of antibodies to multiple islet antigens can predict IDDM at high sensitivity and specificity in families, and such a strategy may also be applicable to identify young individuals in the general population with high disease risk. Since appearance of antibodies to different antigens occurs sequentially rather than simultaneously, accurate assessment of diabetes risk based on the presence of multiple antibodies will require follow-up over a number of years after the first evidence of islet autoimmunity.     

Early development and spreading of autoantibodies to epitopes of IA-2 and their association with progression to type 1 diabetes

Autoimmunity precedes clinical type 1 diabetes, and indicators of maturing autoimmune responses may be useful markers for disease prediction. To study this, epitope maturation of autoantibodies to the related protein tyrosine phosphatase (PTP)-like autoantigens IA-2 and IA-2beta was examined in sequential samples from birth in a cohort of 21 offspring developing multiple islet autoantibodies and a similar cohort of 48 relatives of patients with type 1 diabetes recruited at an older age. Initial reactivity in offspring was heterogeneous against the IA-2 juxtamembrane region (10/21) and PTP domains (13/21), and both specificity and extent of initial IA-2/IA-2beta autoantibodies were associated with HLA class II genotype. Intra-IA-2 epitope spreading and/or intermolecular spreading to IA-2beta epitopes were observed in seven offspring. In contrast, in older relatives, IA-2/IA-2beta Ab reactivity was stable and spreading rare. Development of diabetes in children was associated with the presence of Abs to the IA-2 juxtamembrane region (risk by age 5 yr, 52% vs 0% in those with PTP domain Abs only; p < 0.02), and 5 of 26 relatives who developed diabetes had IA-2 Abs only against the juxtamembrane region. The findings show that autoantibody reactivity to IA-2/IA-2beta is dynamic in the young, show that the juxtamembrane region of IA-2 is an early IA-2 autoantibody target, and suggest that these Abs are a risk factor for development of type 1 diabetes in infancy.     

Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined

IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.     

Epidemiology, clinical aspects, and biology of IDDM patients under age 40 years. Comparison of data from Antwerp with complete ascertainment with data from Belgium with 40% ascertainment. The Belgian Diabetes Registry

OBJECTIVE: To compare the incidence rate of IDDM in the age-groups 0-14 and 15-39 years in Antwerp, Belgium, and to compare demographic, clinical, and biological data from Antwerp IDDM patients with 92% ascertainment with those from a larger Belgian patient group with 40% ascertainment. RESEARCH DESIGN AND METHODS: Incident cases of IDDM were reported by physicians of the Belgian Diabetes Registry and in Antwerp by several other sources. In Antwerp, completeness of ascertainment was calculated by the capture-recapture method. Demographic and clinical data were collected by questionnaire. Blood was sampled for HLA-DQ genotyping and, in new-inset patients, for autoantibodies. RESULTS: In Antwerp, the age- and sex-standardized IDDM incidence rates were similar in both age-groups (0-14 years: 11.8/100,000; 15-39 years: 8.9/100,000). The incidence rate decreased in girls above age 15 years (6.9/100,000; P = 0.003) but not in boys (11.0/100,000). Both in Antwerp and Belgium, IDDM was diagnosed more frequently in the 15-39 years age-group (60% of all cases) than under age 15 years, with a lower prevalence of acute symptoms, ketonuria, high-risk HLA-DQ genotype, and autoantibodies against insulin, islet cells, and IA-2, but with a higher prevalence of GAD65 autoantibodies. CONCLUSIONS: In Antwerp, the incidence rate of IDDM under age 15 years is intermediately high compared with the rates in other European regions. It is similar in the 15-39 years age-group, but with a marked male predominance. Demographic, clinical, and biological data show the same age-dependent heterogeneity as the data collected nationwide, with 40% ascertainment indicating the representativeness of the latter.     

Interactions of age, islet cell antibodies, insulin autoantibodies, and first-phase insulin response in predicting risk of progression to IDDM in ICA+ relatives: the ICARUS data set. Islet Cell Antibody Register Users Study

Many studies have examined the role of age, islet cell antibodies (ICAs), insulin autoantibodies (IAAs), and first-phase insulin responses (FPIRs) to an intravenous glucose tolerance test (IVGTT) as markers of risk of progression to IDDM, but a large data set is required for the analysis of the interactions between these markers. The Islet Cell Antibody Register Users Study (ICARUS) register includes 456 first-degree relatives with ICA levels > or = 5 JDF U confirmed in a reference laboratory, 108 of whom have progressed to IDDM in the course of prospective follow-up. Analysis of this data set confirmed the importance of the loss of FPIR, high ICA titer, coexistence of IAA, and young age in enhancing the risk of progression to the disease. The influence of any given marker of risk is modified by the presence or absence of the other markers. Cox regression analysis performed in a subset of 217 subjects for whom IVGTT, ICA, and IAA data were available showed that risk was most strongly associated with loss of FPIR; IAA and ICA titer contributed equally to the model, while age was also an independent risk determinant.     

Parameters associated with residual insulin secretion during the first year of disease in children and adolescents with Type 1 diabetes mellitus

Factors associated with residual insulin secretion and spontaneous remission in Type 1 diabetic patients are important in the evaluation of treatment aimed at modifying the natural history of Type 1 DM. We investigated the effect of parameters at onset on residual beta cell function in 215 Type 1 DM children and adolescents. Blood gas analysis, HLA, GAD and IA-2 antibodies before the start of insulin treatment were recorded for each patient. Residual C-peptide secretion was assessed by the glucagon test, and parameters of metabolic control (HbA1c and insulin dose U kg(-1) day(-1)) were examined at disease onset and after 3, 6, and 12 months. Residual C-peptide secretion throughout the first year of disease was significantly reduced in patients with disease onset before age 5. Multiple regression analysis showed that low pH at onset showed a significant and independent association with reduced C-peptide at 3 months (p = 0.02) and that the detection of GAD antibodies had a significant independent association with decreased C-peptide secretion at 6 months of follow-up (p = 0.02). Insulin requirement was higher in the youngest patients group and in patients with GAD antibodies. Spontaneous insulin remission (HbA1c <6% and insulin <0.3 U kg(-1) day(-1)) occurred in 22/192 (11%) patients at 3 months of follow-up, in 15/190 (8%) patients at 6 months and in 8/169 (5%) patient at 12 months. Remission was more prevalent in older patients (p = 0.01) and in patients without detectable GAD antibodies: (14/64 vs 8/128, p = 0.001). Sex, IA-2 antibodies and HLA DR were not independently associated with C-peptide secretion, insulin requirement or remission in the first year of Type 1 DM. This study confirms the association of young age, severe acidosis at disease onset, and GAD antibodies with decreased residual beta-cell function and spontaneous remission during the first year of insulin treatment. These factors should be considered in trials evaluating therapies to retain beta-cell function and induce remission at and after disease onset.     

Sensitivity and specificity for detection of islet cell cytoplasmic antibodies using rat pancreatic sections

We determined islet cell cytoplasmic antibodies (ICA) using rat pancreatic sections as a test substrate substitutive for human pancreatic sections by indirect immunofluorescent technique. ICA were measured in sera from 58 patients with insulin-dependent diabetes mellitus (IDDM), 456 with non-insulin-dependent diabetes mellitus (NIDDM), 50 patients with autoimmune diseases, and 110 healthy controls. Seventeen of 58 patients with IDDM showed recent-onset (within 3 months). ICA were also measured in some samples using blood group O human pancreatic sections, and the ICA titers were compared with those measured using rat pancreatic sections. The prevalence of ICA was 55.2% (32/58) in patients with IDDM, 1.5% (7/456) in those with NIDDM, 0% (0/50) in those with autoimmune diseases, and 0.9% (1/110) in the healthy controls. Of the 17 recent-onset IDDM ICA were positive in 14 (82.3%). In comparative study of titers for ICA using rat pancreatic sections or human pancreatic sections, rat pancreatic sections yielded ICA titers as high as human pancreatic sections did. These results demonstrate that ICA assay using rat pancreatic sections was disease-specific, and that antigenicity of the substrate was favorable to ICA. Rat pancreas presents the advantage of greater availability, while providing an identical substrate for ICA. In conclusion, rat pancreatic sections are useful substrate for detecting ICA.     

Age governs gender-dependent islet cell autoreactivity and predicts the clinical course in childhood IDDM

Most IDDM patients temporarily restore some of their beta-cell function following the initiation of insulin therapy. The aim of this study was to analyse the influence of age, gender, metabolic state at diagnosis and presence of autoantibodies (GAD65 antibodies and ICA) on the duration of the clinical partial remission. In total, 149 consecutively diagnosed IDDM children, 0-16 y old (70F, 79M, mean age 9.5 y) were studied. Partial remission was arbitrarily defined as the period when the insulin dose was below 0.5 U/BW 24 h-1 and HbA1c below 7.5%, and occurred in 119/149 patients with a duration between 1 and 38 months. Cox's regression analysis showed that the factors significantly associated with the duration of remission were age, gender, interaction between age and gender, ICA and a high initial HbA1c, whereas GAD65Ab had no influence. Young boys had the shortest remission period, while adolescent boys had the longest, as compared to young and adolescent girls. The ICA-negative patients (n = 42) had a longer remission period (median 9.7 months) than the ICA-positive children (n = 107; 5.0 months; p = 0.0001), regardless of GAD65Ab status. We speculate that the relative insulin resistance, which is more pronounced in pubertal girls than in boys, may be associated with a more rapid increase of exogenous insulin requirement. These findings are important when evaluating the effect of islet cell autoreactivity on the clinical course of IDDM in children.