ICA512(IA-2) epitope specific assays distinguish transient from diabetes associated autoantibodies

ICA512/IA-2, a tyrosine phosphatase-like protein, is one of the major autoantigens in type 1 diabetes. Following phage display characterization of ICA512 autoantigenic epitopes, we developed fluid phase autoantibody radioimmunoassays for a series of ICA512 fragments (F1 [amino acids (aa): 761-964], F2A [aa 256-760], F2B [aa 761-928], and F2C [aa 929-979]). With the hypothesis that 'non-diabetes associated' ICA512 autoantibodies would differ from diabetes associated ICA512 autoantibodies in terms of epitopes recognized, we analyzed ten such serum samples (two from normal control individuals, one from a general population subject with transient ICA512 autoantibodies and seven from relatives of patients with type 1 diabetes who had single transient ICA512 positivity). All but one of the 'non-diabetes associated' ICA512 positive samples (9/10) did not react with Fragment 1 which contains the major antigenic epitopes of the molecule that were recognized by almost all (51/52) ICA512 positive new onset patient samples and pre-diabetic relatives (P< 10(-6)). The great majority of samples (44/52) from the new onset patients and pre-diabetic relatives reacted with at least two fragments and 60% (31/52) with three or more fragments. In contrast, only one sample of the ICA512 'non-diabetes associated' sera reacted with multiple fragments (P< 10(-4)). Our findings suggest that diabetes associated anti-ICA512 autoantibodies react with multiple ICA512 epitopes while non-diabetes associated ICA512 autoantibodies may usually represent reactivity of antibodies with determinants of ICA512 unrelated to type 1 diabetes. The ability to distinguish diabetes associated from non-diabetes associated anti-ICA512 autoantibodies should provide prognostic information and more importantly suggests that even with highly specific radioassays positivity may occur unrelated to type 1 diabetes.     

Antibodies to the Tyrosine Phosphatase-like Protein IA-2 are highly associated with IDDM,but not with Autoimmune Endocrine Diseases or Stiff Man Syndrome

Antibodies to the 40 kD antigen (identified as tyrosine phosphatase IA-2) and glutamate decarboxylase (GAD65) are strongly associated with insulin dependent diabetes mellitus (IDDM). However, antibodies to GAD (GADA) can appear in the absence of IDDM, particularly in stiff man syndrome (SMS) and in some individuals with autoimmune polyendocrine syndrome type II (APS II) and organ specific autoimmune diseases. The aim of this study was to compare the specificity of IA-2 antibodies (IA-2A) and GADA for IDDM by determining their frequency in different patient groups. IA-2 A were present in 64/114 (56%) IDDM patients and 9/19 (47%) APS II patients with IDDM but in only 4/28 (14%) SMS patients. 1/24 (4%) APS II patients without IDDM and 1/113 (0.9%) patients with organ specific autoimmune disease had low level IA-2A. In contrast GADA were present in 77/114 (68%) IDDM patients and 17/19 (89%) APS II patients with IDDM, but also in 25/28 (89%) SMS patients, 5/24 (21%) APS II patients without IDDM and 22/113 (19%) patients with organ specific autoimmune diseases. Furthermore, within the group of new onset IDDM, IA-2A seemed to be associated with ICA and age: 63% of ICA positive IDDM patients had IA-2 A (74% had GADA) increasing to 77% in the group below 20 years of age (69% for GADA).

Our results demonstrate that IA-2A may be more specific for IDDM than GADA, as the latter are also present in patients with SMS, APS II without IDDM and organ specific autoimmune diseases. IA-2A were less frequent in older patients with IDDM than GADA or ICA. A combination of IA-2A and GADA detected 84% of total and 93% of ICA positive IDDM patients.     

Rapid assays for detection of anti-islet autoantibodies: implications for organ donor screening

The purpose of the current study was to develop and evaluate rapid assays for autoantibodies to GAD65 (GAA), ICA512bdc/IA-2 (ICA512AA), and insulin (microIAA, mIAA) as a potential tool for identification of cadaveric pancreas donors who were at high risk for developing diabetes. The study included 154 new onset diabetic, prediabetic, and healthy control subjects. Subjects were evaluated for all three autoantibodies in three separate assays: (1) standard (std) assay with a 24-h or 72-h incubation at 4 degrees C (combined GAA/ICA512AA or mIAA, respectively), (2) rapid assay with 1-h room temperature (RT) incubation, and (3) rapid assay with 2-h RT incubation. The serum samples from 777 organ donors were also evaluated for all three autoantibodies and all the positive samples from standard assay evaluated with the 1-h incubation assay. Simple linear regression analyses revealed excellent correlation between the standard assay and the rapid assays for all three autoantibodies, as follows: (1) GAA: std vs. 1 h (R2=0.85) and std vs. 2 h (R2=0.83), (2) ICA512AA: std vs. 1 h (R2=0.85) and std vs. 2 h (R2=0.84), and (3) mIAA: std vs. 1 h (R2=0.70) and std vs. 2 h (R2=0.64). Comparison of assay correlation rates between subject cohorts revealed no significant differences. Compared to their respective standard assays, the 1-h RT GAA assay missed 3.2% and identified an additional 1.3% of samples, the 1-h RT ICA512AA assay had no discordant samples, and the 1-h RT mIAA assay missed 7.1% and identified an additional 5.8% of samples. We analysed a series of 777 stored serum samples from cadaveric donors. Two of 777 (0.25%) were positive for two autoantibodies (both GAA and ICA512AA) and 23 of 777 (3.0%) one autoantibody (11 IAA; 12 GAA). The rapid analysis for all three autoantibodies could be completed in less than 3 h with comparable concordance rates to the more time-consuming standard assays, making these assays an attractive option for organ donor screening to identify potential pancreata for immunopathogenetic research.     

Mapping of novel autoreactive epitopes of the diabetes-associated autoantigen IA-2

IA-2, a member of the tyrosine phosphatase family, has been identified as a dominant autoantigen in type 1 diabetes. To define humoral IA-2 epitopes, we generated a panel of IA-2 deletion mutants and chimeric proteins using the highly homologous tyrosine phosphatase-like protein IA-2beta. Analysis of autoantibody reactivity in 111 IA-2 antibody positive sera from patients with type 1 diabetes revealed that humoral epitopes cluster to several domains of the intracytoplasmic part of IA-2 [IA-2ic, amino acid (aa) 604-979]. Immunodominant epitopes were found in the first N-terminal 73 amino acids (56% positive), in the middle domain residing between residues 699-874 (45% positive) and the C-terminus depending on the presence of aa 931-979 (at least 37% positive). Competition experiments with overlapping peptides revealed that autoantibody binding towards the N-terminus was dependent on residues 621-628. In the C-terminal domain, two novel conformation-dependent epitopes were identified. The first epitope requires the presence of the C-terminal part of IA-2 (aa 933-979) and an IA-2-specific region between residues 771-932. Reactivity against the second epitope was dependent on intact C-terminal domains as well as residues in the middle (aa 887-932) and N-terminal regions (aa 604-771) which are conserved in IA-2 and IA-2beta. We here defined novel autoantigenic determinants in the N-terminus of IA-2 and characterized conformational epitopes residing in the C-terminal region or spanning from C-terminal residues to the N-terminal domain of IA-2ic. The identification of dominant target regions of diabetes-specific autoantibodies may help to elucidate the molecular mechanisms involved in the autoimmunity towards IA-2.     

Autoantibodies to multiple islet autoantigens in patients with abrupt onset type 1 diabetes and diabetes diagnosed with urinary glucose screening.

It has been reported that there is a heterogeneity in the clinical course of Japanese patients with type 1 diabetes. To elucidate the associations of expression of autoantibodies to multiple islet antigens with age of onset and mode of diagnosis of diabetes in Japanese patients with type 1 diabetes, autoantibodies against the protein tyrosine phosphatase-like molecules ICA512 (IA-2) and phogrin (IA-2beta) (ICA512/phogrin-A), GAD (GADA), insulin (IAA), and islet cell cytoplasm (ICA) were determined in sera from 73 Japanese patients with type 1 diabetes obtained within 14 days of diagnosis. Patients were divided into groups based on the age of onset (10 years, n=49) or the mode of onset (abrupt onset, n=59 and urinary screening identified, n=14). Of 73 new-onset patients with type 1 diabetes, 43 (59%) and 32 (44%) had ICA512A and phogrin-A levels exceeding the 99th percentile of 184 normal control subjects, respectively. Forty-five patients (62%) were positive for either ICA512A or phogrin-A. The frequencies for other autoantibodies were 71% for GADA, 48% for IAA, and 62% for ICA. The frequency of ICA512/phogrin-A was significantly higher in patients with an age of onset less than 10 years (83%) than in patients aged >10 years (51%, P<0.01). The positivity of ICA512/phogrin-A was less in patients whose diabetes was diagnosed by the urine glucose screening test (21%, P<0.001) than in abrupt onset patients (71%). Combined analysis (>/=1 antibody) of GADA, IAA, and ICA512/phogrin-A detected 88% of abrupt onset and 93% of screening-positive patients vs. 70% and 29%, respectively, for ICA (P<0.0005). These results indicate that the expression of ICA512/phogrin-A and cytoplasmic ICA is less in patients identified by urinary glucose testing but indicate that with combined autoantibody testing 90% of patients can be identified independent of the mode of diagnosis.     

Expression of protein tyrosine phosphatase-like molecule ICA512/IA-2 induces growth arrest in yeast cells and transfected mammalian cell lines

The ICA512/IA-2 molecule, a protein with similarity to receptor-type protein tyrosine phosphatases, was discovered during studies to identify autoantigens in Type 1 diabetes. The biological function of ICA512/IA-2 is unknown. We describe striking effects of ICA512/IA-2 on viability and growth of both yeast cells and cultured mammalian cells. In transformed yeast Saccharomyces cerevisiae cells, expression of ICA512/IA-2 induced growth retardation as judged by measurements of optical density and counts of colony-forming units. In contrast, expression of the intracellular domain (amino acids 600-979) of ICA512/IA-2 in yeast or mammalian cells had no such effects. In investigations on apoptosis, expression of ICA512/IA-2 in yeast cells caused loss of plasma membrane asymmetry, but not release of cytochrome c from mitochondria which did occur in a control system after expression of the pro-apoptotic molecule Bax. Possible interactions between ICA512/IA-2 and components of the cytoskeleton were not supported by studies on staining of fixed yeast cells with phalloidin-Texas Red. With transfected mammalian cell lines COS-7 and NIH3T3, expression of ICA512/IA-2 likewise induced growth arrest, with some of the morphological features of apoptosis. Thus obligatory expression of ICA512/IA-2 in eukaryotic cells causes disruption of cellular activities, with growth arrest in yeast and nuclear pycnosis/fragmentation in mammalian cells. A possible explanation is that growth inhibition reflects a part of the presently unknown function of ICA512/IA-2.     

Association Between IA-2 Autoantibody Epitope Specificities and Age of Onset in Japanese Patients with Autoimmune Diabetes

The IA-2 is a major autoantigen of type 1 diabetes belonging to the protein tyrosine phosphatase family. We report on the humoral autoimmunity to an alternatively-spliced variant of IA-2 (IA-2 variant) and autoimmune-mediated diabetes age of onset association with IA-2 autoantibody epitope specificities, in 144 recent-onset patients with type 1 diabetes and 54 GAD autoantibody-positive patients with type 2 diabetes. The cytoplasmic domain of IA-2 (IA-2ic) detected a somewhat greater proportion of patients expressing autoantibodies than IA-2 variant (56%vs. 52% of patients with type 1 diabetes and 17%vs. 9% of GAD autoantibody-positive patients with type 2 diabetes). Conversely, only 1% of IA-2 variant autoantibody-positive patients failed to react to IA-2ic construct. Among 80 patients with type 1 diabetes who were positive for autoantibodies to IA-2ic, 8% recognized the juxtamembrane region (JM, representing amino acids 601-629) only, 64% bound the protein tyrosine phosphatase (PTP)-like domain of IA-2 only, and 29% bound both JM and PTP epitopes. Autoantibodies to the PTP-like domain were prevalent in children and adolescents with type 1 diabetes. The age of disease onset in patients with IA-2JM autoantibodies only, was significantly higher than those in patients reacted with the PTP-like domain of IA-2 (P< 0.02). Among GAD autoantibody-positive patients with type 2 diabetes reacted with IA-2ic, 44% bound the JM region only, and 33% bound epitopes in the PTP-like domain only; 22% had autoantibodies to both regions. The frequency of GAD autoantibody-positive patients with type 2 diabetes positive for autoantibodies to the JM region only, was significantly higher than that in patients with type 1 diabetes (P< 0.01). IA-2PTP autoantibodies were significantly associated with HLA-DR4, while the additional reactivity to IA-2JM was associated with HLA-DR9 allele. These results suggest that autoantibody recognition of IA-2 epitopes in autoimmune diabetes is associated with age of disease onset, which may reflect the intensity of the beta-cell destruction process.     

Radiobinding assay for detecting autoantibodies to single epitopes

Autoantibodies, a hallmark of autoimmune disease, are directed against diverse antigens and epitopes. This diversity aids in characterising disease progression and identifying disease-associated autoantibodies. Here we aimed to develop a sensitive assay to detect and quantify epitope-specific autoantibodies. We generated constructs to integrate known peptide epitopes into the TrxA protein, and used these to in vitro synthesize radio-labelled proteins for a radiobinding assay (RBA). This assay was first validated with an animal model using mice immunized with the MOG(40-55) peptide. Using type 1 diabetes-associated protein tyrosine phosphatase-like autoantigen IA-2 as a human model, we expressed IA-2(609-621), IA-2(619-631), or IA-2(609-631) peptide in the TrxA construct and used the RBA to test sera from 113 patients with type 1 diabetes and 87 controls. Antibodies were detected to the IA-2(609-631) epitope in 37 of 113 patient sera and one control; 17 sera were reactive to the IA-2(609-621) (JM1) epitope, and 16 to the IA-2(619-631) (JM2) epitope. Interestingly, a novel third epitope (JM3) was identified in 7 sera that reacted to IA-2(609-631) but not the JM1 and JM2 sub-specificities. An ELISA using IA-2(609-631) was less sensitive than the RBA, as it detected only some of the JM1 and JM3 antibody positive sera and none of the JM2. Mutagenesis of single IA-2(609-631) amino acids in combination with the RBA showed that antibodies to JM2 and JM3 were highly diverse in their specific residue requirements. Our strategy using in vitro synthesized antigens and the RBA was thus a highly sensitive and versatile method to identify epitopes and quantify epitope-specific antibodies.     

Disease-associated autoantibodies and HLA-DQB1 genotypes in children with newly diagnosed insulin-dependent diabetes mellitus (IDDM). The Childhood Diabetes in Finland Study Group

The possible relation between HLA-DQ genotypes and both frequencies and levels of autoantibodies associated with IDDM was assessed by examining HLA-DQB1 alleles and antibodies to islet cells (ICA), insulin (IAA), glutamic acid decarboxylase (GADA) and the protein tyrosine phosphatase-related IA-2 molecule (IA-2A) in 631 newly diagnosed diabetic children under the age of 15 years. ICA were found in 530 children (84.0%), while close to half of the subjects (n = 307; 48.7%) tested positive for IAA. GADA were detected in 461 index cases (73.1%), with a higher frequency in those older than 10 years (78.9% versus 69.2% in the younger ones; P = 0.006). More than 85% of the children (n = 541; 85.7%) tested positive for IA-2A. Altogether there were only 11 children (1.7%) who had no detectable autoantibodies at diagnosis. There were no differences in the prevalence of ICA or GADA between four groups formed according to their HLA-DQB1 genotype (DQB1*0302/02, *0302/X (X = other than *02), *02/Y (Y = other than *0302) and other DQB1 genotypes). The children with the *0302/X genotype had a higher frequency of IA-2A and IAA than those carrying the *02/Y genotype (93.8% versus 67.3%, P < 0.001; and 49.0% versus 33.6%, P = 0.002, respectively). The children with the *02/Y genotype had the highest GADA levels (median 36.2 relative units (RU) versus 14.9 RU in those with *0302/X; P = 0.005). Serum levels of IA-2A and IAA were increased among subjects carrying the *0302/X genotype (median 76.1 RU versus 1.6 RU, P = 0.001; and 50 nU/ml versus 36 nU/ml, P = 0.004) compared with those positive for *02/Y. Only three out of 11 subjects homozygous for *02 (27.3%) tested positive for IA-2A, and they had particularly low IA-2A (median 0.23 RU versus 47.6 RU in the other subjects; P < 0.001). The distribution of HLA-DQB1 genotypes among autoantibody-negative children was similar to that in the other patients. These results show that DQB1*0302, the most important single IDDM susceptibility allele, is associated with a strong antibody response to IA-2 and insulin, while GAD-specific humoral autoimmunity is linked to the *02 allele, in common with a series of other autoimmune diseases as well as IDDM. We suggest that IA-2A may represent beta cell-specific autoimmunity, while GADA may represent a propensity to general autoimmunity.     

Expression,characterization, processing and immunogenicity of an insulin-dependent diabetes mellitus autoantigen,IA-2, in Sf-9 cells

Autoantibodies to a 64-kD protein and a 40-kD tryptic fragment from pancreatic islets have been detected at high frequency in the sera of patients with insulin-dependent diabetes mellitus (IDDM). IA-2, a newly isolated transmembrane protein tyrosine phosphatase, is a major islet cell autoantigen in IDDM and the precursor of a 40-kD tryptic fragment. To express large quantities of recombinant IA-2 protein and analyse post-translational modifications we expressed full-length human IA-2 in baculovirus-infected Sf-9 cells. IA-2 expression was analysed by Western blot and by immunoprecipitation of ³⁵S-methionine-radiolabelled proteins with rabbit antisera or IDDM sera. A 120-kD IA-2 protein was detected during the early, but not the late, phase of the infection. Pulse-chase experiments showed that the 120-kD protein was processed into fragments of 64 kD and smaller fragments of ≈ 50 kD, 38 kD and 32 kD. The 64-kD fragment appeared as a doublet. Tunicamycin and PNGase F treatment down-shifted the 120-kD protein and the 64-kD doublet into lower molecular weight bands, suggesting that both were glycosylated. Trypsin treatment converted the 120-kD protein and the 64-kD doublet into a 40-kD fragment. Baculovirus-expressed IA-2 was as sensitive or slightly more sensitive than in vitro translated IA-2 in detecting autoantibodies to IA-2: 66% of sera from newly diagnosed IDDM patients reacted with baculovirus-expressed IA-2 compared with 59% of the same sera which reacted with in vitro translated IA-2. It is concluded that baculovirus-expressed IA-2 is a good source of autoantigen and that a number of lower molecular weight fragments with which IDDM autoantibodies react are derived from the 120-kD full-length IA-2 molecule.     

Production of the islet cell antigen ICA69 (p69) with baculovirus expression system: analysis with a solid-phase time-resolved fluorescence method of sera from patients with IDDM and rheumatoid arthritis.

Islet cell antigen 69 (ICA69), previously implicated as an autoantigen in autoimmune insulin-dependent diabetes mellitus (IDDM), was produced using baculovirus-mediated expression in Spodopterafrugiperda (Sf9) insect cells. In these cells the protein was effectively expressed and ICA69 carrying C-terminal histidine-hexapeptide could be efficiently purified using immobilized metal chelate affinity chromatography. Screening of patient and control sera using this protein as an antigen in time-resolved fluoroimmunoassay (TR-FIA) identified 4/50 of patients with IDDM and 6/73 of patients with rheumatoid arthritis (RA) to be positive for ICA69 antibodies. The number of positives did not differ significantly between patients and control subjects but the level of binding was higher in sera from RA patients compared to that of control sera (P = 0.003). The results show that some subjects have specific autoreactive antibodies against the ICA69 protein produced with recombinant technology.     

Autoantibodies to glutamic acid decarboxylase (GAD) detected by an immuno-trapping enzyme activity assay: relation to insulin-dependent diabetes mellitus and islet cell antibodies.

It has recently been proposed that the islet 64,000 Mr protein autoantigen (64K) of insulin-dependent diabetes mellitus (IDDM) is glutamic acid decarboxylase (GAD). We evaluated, by means of a newly developed immunotrapping enzyme activity assay (ITEAA), the prevalence of circulating GAD-autoantibodies (Ab) in a large population of IDDM patients (n = 168), blood donors (n = 87) and non-diabetic autoimmune patients (n = 40). The latter two groups were used as controls. Overall, GAD-Ab were found in 22% of IDDM patients, but in none of the two control groups (P = 0.007). These specificities were invariably associated with islet cell antibodies (ICA) (31.6% in IDDM with ICA vs 0 in IDDM without ICA, P = 0.0001), and this prevalence was higher in sera with high titer ICA (54.5% in IDDM with ICA greater than 80 JDF-units vs 22.6% of IDDM with ICA 5-80 JDF units; P = 0.002). Moreover, GAD-Ab were associated with the female sex (P = 0.002) and the concomitant presence of thyroid and/or gastric antibodies (P = 0.002). No correlation was observed between GAD-Ab and age of the patients, duration of IDDM, or associated non-organ specific antibodies. Our study indicates that GAD-Ab measured by ITEAA are: (1) detected in a proportion of IDDM patients; (2) strongly associated with ICA; (3) preferentially found in IDDM female patients with autoimmune polyendocrine serology; and (4) detected with lower frequency than that reported for 64K-Ab in IDDM.     

CTLA-4 gene polymorphism confers susceptibility to insulin-dependent diabetes mellitus (IDDM) independently from age and from other genetic or immune disease markers

Apart from genes in the HLA complex (IDDM1) and the variable number of tandem repeats in the 5′ region of the insulin gene (INS VNTR, IDDM2), several other loci have been proposed to contribute to IDDM susceptibility. Recently, linkage and association have been shown between the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) gene on chromosome 2q and IDDM. In a registry-based group of 525 recent-onset IDDM patients < 40 years old we investigated the possible interactions of a CTLA-4 gene A-to-G transition polymorphism with age at clinical disease onset and with the presence or absence of established genetic (HLA-DQ, INS VNTR) and immune disease markers (autoantibodies against islet cell cytoplasm (ICA); insulin (IAA); glutamate decarboxylase (GAD65-Ab); IA-2 protein tyrosine phosphatase (IA-2-Ab)) determined within the first week of insulin treatment. In new-onset IDDM patients, G-allele-containing CTLA-4 genotypes (relative risk (RR) = 1.5; 95% confidence interval (CI) = 1.2–2.0; P< 0.005) were not preferentially associated with age at clinical presentation or with the presence of other genetic (HLA-DR3 or DR4 alleles; HLA-DQA1*0301-DQB1*0302 and/or DQA1*0501-DQB1*0201 risk haplotypes; INS VNTR I/I risk genotype) or immune (ICA, IAA, IA-2-Ab, GAD65-Ab) markers of diabetes. For 151 patients, thyrogastric autoantibodies (anti-thyroid peroxidase, anti-thyroid-stimulating hormone (TSH) receptor, anti-parietal cell, anti-intrinsic factor) were determined, but association between CTLA-4 risk genotypes and markers of polyendocrine autoimmunity could not be demonstrated before or after stratification for HLA- or INS-linked risk. In conclusion, the presence of a G-containing CTLA-4 genotype confers a moderate but significant RR for IDDM that is independent of age and genetic or immune disease markers.     

Detection and characterization of ZnT8 autoantibodies could help to screen latent autoimmune diabetes in adult-onset patients with type 2 phenotype

Autoantibodies to zinc transporter 8 (ZnT8A) constitute an additional marker of autoimmune diabetes, complementing those already used in diagnosis support. ZnT8A could also be found in latent autoimmune diabetes of adults (LADA). The aim of this study was to evaluate the prevalence of ZnT8A in adult-onset diabetic patients in Argentinian population. A total of 271 patients diagnosed for diabetes at mean age 53.4 ± 10.9, body mass index ≤ 30, without insulin treatment for the first year of disease, and initially classified as type 2 diabetic patients were tested for ZnT8A using cDNA plasmids encoding the C-terminal domains (aa 268-369) carrying 325Arg, 325Trp, and a dimeric cDNA construct carrying both 325Arg and 325Trp (ZnT8 Arg-Trp325). We also analyzed proinsulin autoantibodies (PAA), glutamic acid decarboxylase autoantibodies (GADA), and protein tyrosine phosphatase IA-2 autoantibodies (IA-2A). A subset of 101 patients was followed during 6 years in order to analyze insulin requirement. Out of the 271 patients, 22.1% presented at least one humoral marker, 2.6% were PAA+, 12.5% were GADA+, 3.3% were IA-2A+, and 10.7% were ZnT8A+. Among the latter, 7.0% were ZnT8A-Arg325, 51.7% were ZnT8A-Trp325, and 62.1% were ZnT8A-Arg-Trp325. Furthermore, the prevalence of autoantibodies in the group of patients treated with insulin (n = 18) was 55.6%. These results demonstrated that a significant proportion of autoimmune adult-onset diabetic patients presented ZnT8A as the only humoral marker. Between them, the higher prevalence was for ZnT8A-Trp325. We suggest that screening for LADA patients, best performed with a minimal set of marker determination, must include at least the screening of GADA and ZnT8A-Arg-Trp325.     

Immunogenetic heterogeneity in type I (insulin-dependent) diabetes among the Tunisian population. HLA-DR antigens and organ-specific autoantibodies (family study)

The frequency of HLA-DR antigens, as well as the prevalence of islet cell insulin autoantibodies and other autoimmunity disorders, were investigated in Tunisian patients with insulin-dependent diabetes mellitus (IDDM) and were compared with family members (sibs) and healthy control subjects. Cytoplasmic islet cell autoantibodies (ICA) were found in 79 of 175 (45.1%) patients with IDDM, in 23 of 126 (18.25%) unaffected first degree relatives of type I diabetes patients and in only two of 146 (1.3%) control subjects. In 79 ICA positive patients with IDDM, 46.8% presented other evidence of autoimmunity by testing for specific autoantibodies. Insulin autoantibodies were found in 86.9% of healthy ICA-positive sibs. A good correlation between HLA-DR3/DR4 heterozygous phenotypes and the presence of ICA in patients with IDDM and their unaffected sibs was observed in the Tunisian population. In fact, this heterozygous phenotype is found in 63.3% of ICA-positive diabetic patients and in 44.4% of ICA-positive unaffected sibs, whereas, HLA-DR3/DR4 antigens were noted in only 22.9% of ICA-negative diabetic patients and in no ICA-negative unaffected sibs. In these studies, we also summarized the distribution of HLA-DR antigens in patients with IDDM who presented autoimmune disorders other than ICA.     

Association between autoantibody markers and subtypes of DR4 and DR4-DQ in Swedish children with insulin-dependent diabetes reveals closer association of tyrosine pyrophosphatase autoimmunity with DR4 than DQ8

Abstract: HLA DQA1*0301-DQB1*0302 (DQ8) and DQA1*0501-DQB1*0201 (DQ2) are positively and DQA1*0102-DQB1*0602 (DQ6) negatively associated with IDDM. In DQA1*0301-DQB1*0302 (DQ8)-positive patients, susceptibility is also mediated by DRB1*0401. The aim of the study was to determine the association between HLA-DR4 and DQ and the presence of GAD65, ICA512, and insulin autoantibodies as well as ICA in 425 Swedish children with IDDM and 367 controls in the age group of 0–15 years. We found that ICA512 autoantibodies were associated primarily with DRB1*0401 and not with DQA1*0301-DQB1*0302 (DQ8). No such hierarchy could be demonstrated for insulin autoantibodies, which were associated with both DQA1*0301-DQB1*0302 (DQ8) and DRB1*0401. GAD65 autoantibodies, known to be closely associated with DQA1*0501-DQB1*0201 (DQ2)-DRB1*0301 haplotype, also showed no preferential association with DQA1*0301-DQB1*0302 (DQ8) versus DRB1*04. These results suggest that the immune response to different β-cell autoantigens may be mediated via HLA class II molecules from different loci. Design of the antigen-specific immuno-intervention trials should take into account these HLA-DR and DQ subtype associations.     

Humoral beta-cell autoimmunity is rare in patients with the congenital rubella syndrome

The congenital rubella syndrome (CRS) is associated with increased risk for diabetes and thyroid disease. However, the mechanisms by which the rubella virus may cause these diseases are poorly characterized. Previous studies were carried out before modern immunological methods were available. The present study aimed at evaluating whether autoimmune mechanisms are involved in the pathogenesis by analysing antibodies to biochemically characterized autoantigens. The incidence of clinical diabetes, thyroid disease, coeliac disease and related antibodies (islet cell antibodies, ICA; insulin autoantibodies, IAA; antibodies to the tyrosine phosphatase related IA-2 molecule, IA-2 A and glutamic acid decarboxylase, GADA; thyroid peroxidase, TPO; tissue transglutaminase, TTGA; and gliadin, AGA) and HLA risk genotypes were analysed in 37 subjects affected by or exposed to rubella during fetal life (mean age 22.5 years). One patient had diabetes and four patients had clinical hypothyroidism at the time of the examination. ICA, IAA, GADA or IA-2 A were not detected in any of the patients, while five patients tested positive for TPO antibodies. Coeliac disease or TTGA were not observed. Eight patients carried the HLA-DR3-associated HLA-DQB1*02-DQA1*05 haplotype. These results provide no evidence of an increased frequency of markers for humoral beta-cell autoimmunity in patients with CRS suggesting that diabetes in CRS may be caused by other than autoimmune mechanisms.     

In vitro secretion of interleukin 2 and expression of IL-2 receptor in peripheral blood lymphocytes in high risk of insulin-dependent diabetes mellitus subjects.

Interleukin 2 (IL-2)--a Th1 lymphocyte-derived cytokine is at present considered to play an important role in the etiopathogenesis of insulin-dependent diabetes mellitus. In the previous studies increased, decreased and unchanged IL-2 levels in patients with recent onset of insulin-dependent diabetes mellitus (IDDM) were found. These differences could be a result of different metabolic status or/and a different stage of the autoimmune process. The aim of our study was to estimate in vitro secretion of IL-2 and CD25 antigen expression by the peripheral blood T lymphocytes in subjects at the preclinical stage of IDDM (prediabetes), but still without metabolic disturbances. In 27 first degree relatives of IDDM patients with antibodies against different pancreatic islet cell antigens (ICA, GADA, IAA, IA-2) CD25 antigen expression on peripheral blood lymphocytes T was measured by flow cytometry and IL-2 concentration in supernatants of 48 and 72 h cultures of peripheral whole blood with 10 microg/ml PHA was estimated by ELISA. The control group was comprised of 34 age and sex-matched healthy volunteers. In the studied high risk IDDM subjects the decreased CD25 expression in peripheral CD4+ lymphocytes T and a negative correlation between the percentage of CD25+ cells and islet cell antibodies (ICA) titres was observed. No differences in IL-2 levels in supernatants of 48 h and 72 h blood cultures was found in subjects with single antibody (ICA+) in comparison to healthy controls. A significant increase of IL-2 secretion at 72 h of PHA stimulation was shown in first degree relatives of IDDM patients with a combination of 3 or more antipancreatic-B cell antibodies. There were also a significant negative correlation between glutamic acid decarboxylase antibodies (GADA) titres and IL-2 levels in 72 h of culture. The present study suggests the involvement of IL-2 in the pathogenesis of IDDM. The estimation of CD25 antigen expression in the peripheral blood lymphocytes could be an additional immunological marker of identification of subjects in prediabetes.     

Pancreatic autoantibodies in Tunisian children with newly diagnosed type 1 diabetes

We analyzed 86 children with newly diagnosed type 1 diabetes for antibodies to islet cells (ICA), glutamic acid decarboxylase (GADA), second-islet antigen (IA-2A), and insulin (IAA) in order to evaluate the prevalence of immune-mediated type 1 diabetes, as well as to recognize which autoantibody combination is more frequently associated with the disease. A positive result for one or more diabetes-related antibodies evaluated was found in 78 children (90.7%). With regard to single autoantibody testing, ICA were found to be positive in 49 patients (57%), GADA in 56 (65.1%), IA-2A in 37 (43%), and IAA in 43 (50%) patients. Combining the determination of at least two autoantibodies, GADA and/or IAA were better detectable than other antibody combination, being positive in 70 patients (81.4%). GADA and IA-2A represent also a useful screening combination; being positive in 65 patients (75.6%). Our data indicate that the vast majority of cases of type 1 diabetes in children may be considered as immune-mediated and that multiple autoantibody analysis improves identification of the disease.