Cytoplasmic Islet Cell Antibodies Research Articles

Islet cell antibodies in individuals at increased risk for IDDM.

Islet cell cytoplasmic antibodies (ICA), islet cell surface ( ICSA ) antibodies, HLA phenotypes, glucose tolerance, insulin secretion, and insulin sensitivity were studied in 16 twins of insulin-dependent diabetics as well as in 21 subjects with impaired glucose tolerance (IGT). 60% of the identical twins and 40% of the non-identical twins were ICSA -positive. The prevalence of ICSA in control persons was only 5%. ICA were found in all identical twins and in half of the non-identical twins. However, ICSA and ICA results were concordant in only 46% of the whole group of twins. There was no correlation between ICSA and either insulin secretion or insulin sensitivity. In the IGT subjects exhibiting low and normal insulin responses ICSA were observed in 67% and 23%, respectively. A high proportion of twins, but not of IGT subjects, had HLA DR3 or DR4 antigens which seem to confer genetic susceptibility to the development of IDDM. In the majority of DR3/DR4 twins, ICSA were also present. This might support the hypothesis of genetically-based autoimmunity, although the precise relationship between HLA and islet cell antibodies has to be clarified in a prospective study.

Inhibition of glucagon release of isolated islets of Langerhans by monoclonal antibodies.

The presence of islet cell cytoplasmic antibodies (ICA) and islet cell surface antibodies (ICSA) at the time of diagnosis of type 1 (insulin-dependent) diabetes mellitus has been taken as evidence that autoimmune mechanisms are involved in the pathogenesis of the disease. The demonstration that ICSA in the presence of complement are preferentially lytic for beta-cells may be important in defining the role of these autoantibodies in the pathogenesis of type 1 diabetes. Because of the polyclonality of the immune response, the ICA and ICSA molecules of diabetic patient vary enormously in their binding parameters. For this reason we have generated monoclonal antibodies (MC-Ab) to islet cell antigens. In this study we investigate the effect of the two MC-Ab K28 A1 and K28 D6 resulted from the same fusion of the P3-X63-Ag8 murine myeloma cell line with the spleen cells of a Balb/c mouse immunized with rat islet cells on the hormone release of isolated rat islet in co-culture with the antibody-secreting hybridomas. The MC-Ab K28 D6 binds to both islet cell cytoplasmic and surface antigens, the K28 A1 is only reactive with cytoplasmic antigens. Surprisingly, in contrast to the monoclonal antibody K28 A1, K28 D6 enhanced the glucagon content and diminished the insulin secretion of the islets. Either the K28 D6 is directed to an epitope occurring on the beta- as well as alpha-cells or the antibody-mediated inhibition of the glucagon release results in a significantly reduced insulin secretion.

Generation and partial characterization of monoclonal antibodies reactive with islet cell antigens.

Islet cell antibodies have been detected in more than 60% of newly diagnosed type I diabetics. Their pathogenetic role is still unclear. We have generated monoclonal antibodies (mc-ab) reactive with islet cell antigens by fusing mouse myeloma cells with spleen cells from Balb/c mice immunized with pancreatic islet cells. Hybridomas producing islet cell surface antibodies (ICSA) were detected by indirect immunofluorescence on viable cells from rat islets or rat insulinoma. Cytoplasmic islet cell antibodies (ICA) were detected by indirect immunofluorescence on Bouin-fixed sections of mouse pancreas. The ICSA- and/or ICA-producing hybridomas were cloned twice by limiting dilution. This paper describes six different mc-ab. All hybrid cell lines obtained produced IgM antibodies. Four of them mediate complement-dependent cytotoxicity to viable rat islet cells. In the present study the heterogeneity of circulating ICSA is demonstrated. Also, a monoclonal beta cell surface autoantibody K56aF3 was produced by fusion of spleen cells from a mouse treated with sub-diabetogenic doses of streptozotocin in combination with complete Freund's adjuvant. It was cytotoxic against islet cells up to a dilution of 1:1,000 and it could inhibit the insulin secretion from neonatal rat islets cultured in RPMI 1640 as stimulated by glucose or by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine common with glucose. The latter effect was reversible as indicated by the recovery of insulin secretion in a subsequent culture period without mc-ab. These results suggest that circulating ICSA in type I diabetics may alter beta cell function and thereby contribute to the pathogenesis of type I diabetes.

Islet Cell Antibodies and Viral Infections

The sera of 127 non-diabetic children after mumps-infection were investigated for the presence of islet cell antibodies and islet cell surface antibodies. The study also included 4 children who developed diabetes mellitus shortly after an active mumps vaccination. 21 of the non-diabetic children and four of the vaccinated children exhibited islet cell cytoplasmic antibodies. Islet cell surface antibodies were observed more frequently, namely in 43 out of 68 patients studied after mumps infection and in 32 out of 44 patients studied after different viral diseases. With one exception, none of the mumps-infected children and none of the other viral infected patients developed diabetes mellitus.

Longitudinal study of islet cell antibodies and insulin autoantibodies and development of diabetes in non-obese diabetic (NOD) mice

We have previously shown the presence of circulating islet cell cytoplasmic antibodies (ICA) and insulin autoantibodies (IAA) in the NOD mouse before onset of insulin-dependent diabetes mellitus (IDDM). Here we have determined the levels of the two autoantibodies in 28 female NOD mice longitudinally from approximately day 40 to day 250, to examine their ontogeny, association and predictive value for diabetes. All animals (11 diabetic, 17 non-diabetic) showed varying levels of ICA at some stage, while IAA activity was found in 21 out of 28 mice. Expression of both the markers was seen in more than half of the animals by day 60, with higher levels and rates occurring subsequently in both diabetic and non-diabetic groups. The expression of ICA did not always correlate with that of IAA. There was no apparent difference in the ontogeny of ICA and IAA between the two groups. During the study period the number of animals with ICA was similar in the two groups, while the number of those with IAA was higher in the diabetic animals. In this group declining and rising levels of ICA were seen just before clinical diabetes with frequent peaks of IAA. In the same animals, eight out of 11 mice showed co-expression of high levels of both markers either intermittently or persistently prior to onset, whereas only one non-diabetic animal showed this. We conclude that the ontogeny and serum level of ICA or IAA alone could not be used to predict the clinical onset of diabetes in these animals. However, co-expression of high levels of both markers prior to onset may suggest a strong predisposition to clinical diabetes. This may have relevance to attempts to predict the onset of IDDM in humans who have one or both of these immunological markers.

Autoimmune diagnostics in diabetes mellitus

Type 1 diabetes results from a specific destruction of the insulin-producing beta-cells of the pancreas. The disease is characterized by the appearance of specific autoantibodies against islet cell antigens. Autoantibodies to insulin, glutamic acid decarboxylase, tyrosine phosphatase IA-2 and cytoplasmic islet cell antibodies are useful markers for the differential diagnosis of type 1 diabetes when clinical and metabolic criteria alone do not allow definite classification. Autoimmune diagnostics is of particular importance in adults to discriminate between type 1 and type 2 diabetes and to assess the diagnosis of latent autoimmune diabetes in adults.

Cytoplasmic islet cell antibodies remain valuable in defining risk of progression to type 1 diabetes in subjects with other islet autoantibodies

The discovery of islet cell antibodies (ICAs) was the prelude to the understanding that type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease. The issue regarding whether or not the measurement of ICAs should be completely replaced by biochemical markers detecting islet autoantibodies (AAs) for the prediction of T1DM has been the subject of endless international debates. In light of this controversy, we assessed the current role of ICAs as a predictive marker for T1DM progression. We examined a cohort of 1484 first-degree relatives (FDRs) of T1DM probands from the Children's Hospital of Pittsburgh Registry. These relatives were consecutively enrolled between 1979 through 1984 and followed up to 22 yr. Serum obtained at the time of enrollment was assayed for ICAs, glutamic acid decarboxylase (GAD)65, insulin A (IA)-2 AA, and insulin AAs (IAAs). In FDRs who had ICAs in addition to GAD65 and IA-2 AAs, the cumulative risk of developing insulin-requiring diabetes was 80% at 6.7 yr of follow-up, whereas this risk in those with GAD65 and IA-2 AAs without ICAs was only 14% at 10 yr of follow-up (log rank: P < 0.00001). Cox regression analysis showed that diabetes risk was significantly associated with the presence of ICAs in both subjects with low titer and high titer GAD65 and IA-2 AAs. The addition of IAAs in GAD65 and IA-2 AA-positive relatives did not increase the cumulative risk for conversion to insulin-treated diabetes. We provide evidence that a subgroup of ICAs predicts a more rapid progression to insulin-requiring diabetes in GAD65 and IA-2 AA-positive relatives and should remain part of the assessment of T1DM risk for intervention trials. In addition, these findings provide impetus for efforts to identify a novel islet autoantigen(s) reactive with this ICA subset.

Islet antibodies and remaining beta-cell function 8 years after diagnosis of diabetes in young adults: a prospective follow-up of the nationwide Diabetes Incidence Study in Sweden.

To establish the prevalence of remaining beta-cell function 8 years after diagnosis of diabetes in young adults and relate the findings to islet antibodies at diagnosis and 8 years later. Population-based cohort study. Nationwide from all Departments of Medicine and Endocrinology in Sweden. A total of 312 young (15-34 years old) adults diagnosed with diabetes during 1987-88. Plasma connecting peptide (C-peptide) 8 years after diagnosis. Preserved beta-cell function was defined as measurable C-peptide levels. Three islet antibodies - cytoplasmic islet cell antibodies (ICA), glutamic acid decarboxylase antibodies and tyrosine phosphatase antibodies - were measured. Amongst 269 islet antibody positives (ab+) at diagnosis, preserved beta-cell function was found in 16% (42/269) 8 years later and these patients had a higher body mass index (median 22.7 and 20.5 kg m-2, respectively; P = 0.0003), an increased frequency of one islet antibody (50 and 24%, respectively; P = 0.001), and a lower prevalence of ICA (55 and 6%, respectively; P = 0.007) at diagnosis compared with ab+ without remaining beta-cell function. Amongst the 241 patients without detectable beta-cell function at follow-up, 14 lacked islet antibodies, both at diagnosis and at follow-up. Sixteen per cent of patients with autoimmune type 1 diabetes had remaining beta-cell function 8 years after diagnosis whereas 5.8% with beta-cell failure lacked islet autoimmunity, both at diagnosis and at follow-up.

An association of autoantibody status and serum cytokine levels in type 1 diabetes.

At onset of type 1 diabetes, the islet autoantibody status of patients has been reported to predict progression of the disease. We therefore tested the hypothesis that the systemic immunoregulatory balance, as defined by levels of circulating cytokines and chemokines, is associated with islet autoantibody status. In 50 patients with recent-onset type 1 diabetes, antibodies to GAD and insulinoma-associated antigen 2 (IA-2) were analyzed by radioimmunoassay; cytoplasmic islet cell antibodies were determined by indirect immunofluorescence. Cytokine and chemokine concentrations were measured by rigidly evaluated double antibody enzyme-linked immunosorbent assay. Of four classically defined Th1/Th2 cytokines (gamma-interferon, interleukin [IL]-5, IL-10, IL-13), none showed an association with multiple autoantibody positivity. Of six mediators mainly produced by innate immunity cells, three were associated with multiple autoantibody status (IL-18 increased, MIF and MCP-1 decreased) and three were unaffected (IL-12, MIP-1beta, IP-10). GAD and/or IA-2 antibody titers negatively correlated with systemic concentrations of MIF, MIP-1beta, and IL-12. Combining the data of several cytokine and chemokine levels made it possible to predict islet antibody positivity in individual patients with 85% sensitivity and 94% specificity. These data suggest a close association of islet antibody status with systemic immunoregulation in type 1 diabetes.

A mathematical model for pattern of change in beta-cell reserve and factors affecting residual reserve within the first 2 years of type 1 diabetes.

The aim of this study was to investigate the effects of age, duration of diabetes, sex and ICA (Islet cell cytoplasmic antibody) on beta-cell reserves and to develop a model within the first 2 years of Type 1 diabetes. Beta-cell reserve is evaluated as fasting (FCp) and 1 mg i.v. glucagon stimulated C-peptide (SCp) levels in 58 Type 1 diabetics and in 12 normoglycemic subjects. Patients were divided into 3 groups according to duration of diabetes: Group I (2.5+/-0.3 weeks), Group II (13.4+/-1.2 months) and Group III (24.2+/-1.8 months). FCp/SCp level in nmol/l (mean+/-SE) were as follows. Group I: 0.21+/-0.02/0.38+/-0.04, Group II: 0.15+/-0.01/0.27+/-0.02, Group III: 0.07+/-0.01/0.11+/-0.02, CONTROL GROUP: 0.42+/-0.09/1.29+/-0.13. The scatter plots of C-peptide levels vs time in all the diabetic patients fitted in to a 4th-order polynomial regression (R: 0.96-0.98). Age was strongly correlated with FCp (rs: 0.46, p<0.05) and ICA positivity affected Cp-levels negatively (p>0.05). In conclusion, as the duration of diabetes increases, response time to glucagon prolongs and amplitude of it shortens. Duration of diabetes of less than 2 weeks, feminity, puberty and ICA positivity affect beta-cell reserve negatively, conversely, masculinity, post-puberty, older age and ICA negativity affect the reserve positively. The dynamics of C-peptide response to glucagon follow a mathematical model and Type 1 diabetes causes a decrease not only in the amplitude of the response but also in the duration of the response.

Progression to insulin-requiring diabetes in seronegative prediabetic subjects: the role of two HLA-DQ high-risk haplotypes.

Most Caucasians with Type I (insulin-dependent) diabetes mellitus develop an autoimmune form of diabetes known as Type IA diabetes, based on the presence of humoral responses to islet autoantigens. Alleles at the HLA locus account for the strongest susceptibility to this form of diabetes, which requires insulin therapy. Because a number of patients who develop insulin-requiring diabetes are islet autoantibody negative, the HLA class II haplotypes, DQA1*0501-DQB1*0201 and DQA1*0301-DQB1*0302, were evaluated to assess whether they are an independent risk factor for progression to insulin requirement in first-degree relatives of Type I diabetic patients. Both HLA-DQ genotyping and islet cell autoantibody assessment (insulin, GAD65, IA-2 autoantibodies and cytoplasmic islet cell antibodies) were evaluated prospectively in 74 relatives of Type I diabetic patients who developed diabetes treated with insulin (prediabetics) and in 426 control subjects who did not develop insulin-requiring diabetes. Based on the presence of DQA1*0501-DQB1*0201 and/or DQA1*0301-DQB1*0302, the number of HLA-DQ high-risk haplotypes was assigned as 0, 1 or 2. A higher prevalence of 2 HLA-DQ high-risk haplotypes was present in seronegative prediabetic subjects as compared to non-diabetic autoantibody negative first-degree relatives (33.3 % vs 10.1 % respectively; p < 0.05). Moreover, in seronegative relatives who developed insulin-requiring diabetes, the presence of 2 HLA-DQ high-risk haplotypes conferred an increased cumulative risk of developing insulin requirement of 27 % at 12.5 years of follow-up, compared to a risk of 6 % for non-diabetic relatives who were antibody-negative and had 0 or 1 HLA-DQ high-risk haplotypes (Log rank p = 0.01). These data provide evidence for a phenotype, which is associated with the absence of conventional islet autoantibodies at initial screening, while usually remaining seronegative, and the presence of 2 HLA-DQ high-risk haplotypes with progression to clinical Type I diabetes after a prolonged follow-up. Given the fact that in humans the highest risk-conferring locus associated and linked to the disease is the HLA cluster, and that HLA-DQ molecules play a key role in the development of autoimmune diabetes, our observations imply that as yet unidentified immunologic abnormalities could well exist in seronegative relatives at risk of developing clinical diabetes and carrying 2 HLA-DQ high-risk haplotypes.

Prevalence and fate of type 1 diabetes-associated autoantibodies in cord blood samples from newborn infants of non-diabetic mothers.

Cord blood samples were collected from 1002 consecutive births at Turku University Hospital to study the prevalence and fate of type 1 diabetes-associated autoantibodies in newborn infants of unaffected mothers. The samples were analysed for cytoplasmic islet cell antibodies (ICA), autoantibodies to the 65 kD isoform of glutamic acid decarboxylase (GADA), autoantibodies to the protein tyrosine phosphatase related IA-2 antigen (IA-2A), insulin autoantibodies (IAA) and HLA DQB1 genotypes. ICA were detected in 27 cord blood samples (2.7%), with a median of 6 (range 4-34) JDF units. GADA were found to be positive (> or =6.6 RU) in six samples (0.6%), with a median of 66 (range 19-125) RU. IA-2A (> or =0.43 RU) were observed in three samples (0.3%), with a median of 1.3 (range 0.8-57) RU, while only one cord blood sample (0.1%) tested positive for IAA (> or =1.56 nU/ml) with a value of 5.4 RU. Maternal or gestational age, sex and birth weight of the infant were not related to antibody prevalence or titres. Altogether there were 29 infants with antibody positivity in their cord blood (2.9%). Five of these (0.5%) tested positive for two antibodies (ICA and GADA), and one was positive for all four antibodies measured. All nine infants from whom follow-up samples were available became antibody negative by the age of 15 months, and in all but one case inverse seroconversion occurred by the age of 9 months. Around 3% of infants of non-diabetic mothers in Finland have diabetes-associated autoantibodies at birth, and these antibodies disappear at the latest by the age of 15 months.

Expression of GAD65 and islet cell antibody (ICA512) autoantibodies among cytoplasmic ICA+ relatives is associated with eligibility for the Diabetes Prevention Trial-Type 1.

More than 71,000 relatives of type 1 diabetic patients have been screened for cytoplasmic islet cell antibodies (ICAs), GAD65 autoantibodies (GAAs), and ICA512 autoantibodies (ICA512AAs). Among those 71,148 relatives, 2,448 were cytoplasmic ICA+, and the remainder were ICA-. Of the ICA+ group, 1,229 (50.2%) were positive for GAAs and/or ICA512AAs. Among ICA- relatives, 1,897 (2.76%) were positive for GAAs and/or ICA512AAs. Given the large number of relatives positive for cytoplasmic ICA and negative for "biochemically" determined autoantibodies, and the converse, we analyzed the proportion of ICA+ relatives found eligible to participate in the intervention phase of Diabetes Prevention Trial-Type 1 (DPT-1). To be eligible for the parenteral insulin DPT-1 trial, a relative had to have first-phase insulin secretion below the 1st percentile of cut-points (for parents) or below the 10th percentile (for siblings and offspring). To be eligible for the oral insulin trial, a relative had to have first-phase insulin secretion above cut-points (>1st percentile for parents, >10th percentile for siblings/offspring) and be positive for anti-insulin autoantibodies. For both trials, DQB1*0602 was an exclusion criteria, cytoplasmic ICA positivity had to be confirmed, and an oral glucose tolerance test had to result in nondiabetic levels. Of 572 relatives found to be eligible for trial entry, 442 (77.3%) were positive for GAAs and/or ICA512AAs, although overall only 50.2% of ICA+ relatives were positive for GAAs and/or ICA512AAs. The positive predictive value for trial eligibility for ICA+ relatives with GAAs or ICA512AAs who completed staging was 51.0%. In contrast, only 11.9% of ICA+ but GAA- and ICA512AA- relatives were found to be eligible by DPT criteria for trial entry. Positivity for biochemically determined autoantibodies among cytoplasmic antibody-positive relatives is associated with eligibility for the DPT-1 study.

Postnatal elimination of transplacentally acquired disease-associated antibodies in infants born to families with type 1 diabetes. The Finnish TRIGR Study Group. Trial to Reduce IDDM in the Genetically at Risk.

The elimination of maternally acquired, diabetes-associated antibodies from the peripheral circulation of infants was studied in a population of 47 mothers and their newborn infants from families in which at least 1 first degree relative had type 1 diabetes. Blood samples were taken from the placental cord; from the infant at follow-up visits at the ages of 3, 6, 9, 12, 18, and 24 months; and from the mother at the time of delivery. The samples were analyzed for cytoplasmic islet cell antibodies (ICA), insulin antibodies (IA), autoantibodies to the 65-kDa isoform of glutamic acid decarboxylase (GADA), and autoantibodies to the protein tyrosine phosphatase-related IA-2 antigen (IA-2A). The mean elimination times for ICA, IA, GADA, and IA-2A were 3.1, 3.1, 4.5, and 4.3 months (P = NS), respectively. The initial levels of IA, GADA, and IA-2A in the cord blood correlated closely with the elimination time (r(s) = 0:84-0.91; P < 0.001). The mean proportions of ICA, IA, GADA, and IA-2A still detectable were 18%, 21%, 30%, and 20%, respectively, at 3 months; 2.2%, 14%, 10%, and 6% at 6 months; and 0.3%, 15%, 2.3%, and 5.1% at 9 months. One infant still tested positive for GADA at the age of 12 months, whereas all of the other antibodies had been eliminated by that age. When observing the natural history of beta-cell autoimmunity or when screening for secondary prevention in young children, cross-sectional autoantibody analyses do not provide sufficient information. Repeated testing is to be recommended in young children. In infancy, increasing antibody levels most likely reflect de novo synthesis of diabetes-associated autoantibodies.

Autoantibodies to ICA12 (SOX-13) are not specific for Type I diabetes.

The majority of patients with Type I (insulin-dependent) diabetes mellitus has autoantibodies to insulin, glutamic acid decarboxylase and the tyrosine phosphatase-like protein IA-2. Some patients with Type I diabetes, in particular with adult onset diabetes, have, however, only cytoplasmic islet cell antibodies that could be directed to as yet unknown beta cell autoantigens. Recently, one potential antigen, ICA12, was identified as the high mobility group (HMG) box transcription factor SOX-13. Our aim was to evaluate the diagnostic sensitivity and specificity of autoantibodies to SOX-13 for autoimmune diabetes. Full-length SOX-13 was cloned from human brain mRNA, expressed by vitro transcription/ translation and used to detect autoantibodies by immunoprecipitation and radioligand assay in patients suffering from autoimmune diabetes or non-organ-specific autoimmune diseases. We found SOX-13 antibodies to be present in 11 of 125 (8.8%) patients with newly diagnosed Type I diabetes and in 3 of 43 (7.0%) patients with latent autoimmune diabetes in adults. There was no association with age, sex and a particular pattern of diabetes-specific autoantibodies. Similar frequencies of SOX-13 antibodies were found in 84 patients with rheumatic diseases (4.0-11.4%) and 211 healthy control subjects (5.2 %). Our data indicate that SOX-13 represents a minor target of autoantibodies in patients with autoimmune diabetes. Because SOX-13 antibodies were found not to be disease-specific, we assume they are not helpful in improving the diagnosis and prediction of Type I diabetes.

Use of an islet cell antibody assay to identify type 1 diabetic patients with rapid decrease in C-peptide levels after clinical onset. Belgian Diabetes Registry.

To investigate whether the presence of antibody markers at diagnosis could help predict the rapid decrease in residual beta-cell function noted in some, but not all, patients with recent-onset type 1 diabetes. We measured random C-peptide levels (radioimmunoassay); islet cell cytoplasmic antibodies (ICA) (indirect immunofluorescence); and antibodies against IA-2 protein, 65-kDa glutamate decarboxylase, and insulin (liquid-phase radiobinding assays) in 172 patients <40 years of age with type 1 diabetes. The patients had been consecutively recruited at diagnosis by the Belgian Diabetes Registry and were followed for 2 years. Two years after diagnosis, random C-peptide levels had decreased significantly (P < 0.001) in ICA+ patients but not in ICA- patients. C-peptide values <50 pmol/ were noted in 88% of patients diagnosed before 7 years of age, in 45% of patients diagnosed between ages 7 and 15 years, and in 29% of patients diagnosed after 15 years of age (P < 0.001). In cases of clinical onset before age 15 years, a rapid decline in random C-peptide values was observed almost exclusively in patients with high-titer ICA (> or =50 Juvenile Diabetes Foundation [JDF] units) at diagnosis (69 vs. 17% in patients with lower ICA titers, P < 0.001). In patients diagnosed after 15 years of age, 36% of patients with ICA titers > or =12JDF units developed low C-peptide levels compared with 14% of patients with ICA titers < 12 JDF units (P < 0.03). Multivariate analysis confirmed that C-peptide levels after 2 years were inversely correlated with ICA levels (P < 0.001) and to a lesser degree positively correlated with age at diagnosis (P < 0.02), regardless of the levels or number of molecular autoantibodies. Young age at diagnosis and high-titer ICA identify a group of type 1 diabetic patients at high risk of rapidly losing residual beta-cell function. Using these selection criteria, it is possible to better target beta-cell-preserving interventions to patients with or without such rapid progression, depending on the nature of the tested substance. The ICA assay measures clinically relevant antibodies not detected in antibody assays that use recombinant human autoantigens for substrate.

Role of islet autoimmunity in the aetiology of different clinical subtypes of diabetes mellitus in young north Indians

To determine the role of islet autoimmunity in the aetiology of different clinical subtypes of diabetes mellitus in young north Indian patients by measuring islet autoantibodies. In a cross-sectional study, 145 young patients with diabetes (onset < 30 years) were subdivided into the following categories: Type 1 diabetes (n = 83), malnutrition-modulated diabetes mellitus (MMDM, n = 31) and fibro-calculous pancreatic diabetes (FCPD, n = 31). MMDM subjects presented with emaciation and severe insulin-requiring but ketosis-resistant diabetes, while FCPD was associated with idiopathic chronic calcific pancreatitis. Antibodies to glutamic acid decarboxylase (GADA) and IA-2 (IA-2 A) were detected by immunoprecipitation of 35S-labelled recombinant antigens and cytoplasmic islet cell antibody (ICA) by indirect immunofluorescence. GADA were present in a significant proportion (23%) of patients with MMDM. In contrast, IA-2 A was increased only among patients with Type 1 diabetes (22%), but not MMDM (3%, P < 0.05). Among patients with a duration of diabetes < 2 years, GADA and/or IA-2 A were found in 61% of Type 1 diabetic and 37% of MMDM patients (P < 0.01). MMDM patients who were positive for GADA had a shorter duration of diabetes, but did not differ in their age at onset of diabetes, body mass index, fasting plasma C-peptide, or frequency of thyroid microsomal and parietal cell antibodies. FCPD subjects had the lowest prevalence of autoantibodies: IA-2 and ICA were absent, while GADA were present in 7% (P < 0.05 vs. Type 1 diabetes). GADA, though not IA-2 A, were present in a substantial proportion of patients with the MMDM variant of diabetes, suggesting that islet autoimmunity may play a role in its pathogenesis. In contrast, none of the islet antibodies was increased in subjects with FCPD, making it likely that it is a secondary type of diabetes.

Circulating antibodies against an exocrine pancreatic enzyme in type 1 diabetes.

In this article, we report the identification of a new autoantigen in type 1 diabetes originating from the exocrine pancreas. This antigen is a pancreatic enzyme termed bile salt-dependent lipase (BSDL). We show that antibodies present in the sera of newly diagnosed type 1 diabetic patients recognize BSDL and more specifically the COOH-terminal mucin-like region of the protein. Therefore, we engineered the COOH-terminal peptide of BSDL and demonstrated that autoreactivity was linked to specific glycosylation sites by at least two glycosyltransferases: the Core 2 beta(1-6)N-acetylglucosaminyltransferase and the alpha(1-3) fucosyltransferase FUT7. We next examined the prevalence of circulating anti-BSDL antibodies in type 1 diabetic patients and found 73.5% positivity (25 sera among 34 patients tested) at onset, whereas only 8.4% of normal individuals (7 of 83) were positive. Within a cohort of first-degree relatives of diabetic patients followed prospectively until development of diabetes, 6 of 19 (31.6%) were also positive. Interestingly, two prediabetic individuals were already positive for anti-BSDL antibodies (Abs), while islet cell cytoplasmic Abs and antibodies to GAD65, IA-2, and insulin were not detected. Anti-BSDL autoantibodies were weakly or not detected in patients suffering from pancreatitis or pancreatic adenocarcinoma or in patients with Graves' disease. Although autoreactivity to BSDL in prediabetic and newly diagnosed diabetic patients might reflect cross-reactivity, our results strongly suggest that in addition to pancreatic beta-cells, acinar cells may be also affected in type 1 diabetes.

High frequency of diabetes-specific autoantibodies in parents of children with type 1 diabetes. DENIS study group.

Strategies to identify subjects at risk for type 1 diabetes are largely based on the detection of autoantibodies directed to various beta cell autoantigens. Most previous studies only comprise siblings and children of patients with type 1 diabetes; only scare data are available on the antibody profile in older relatives. In this study, we examined the prevalence of cytoplasmic islet cell antibodies (ICA), antibodies to glutamic acid decarboxylase (GADA), antibodies to the protein tyrosine phosphatase IA-2 (IA-2A) and IA-2beta (IA-2betaA) in 531 unaffected parents of patients with type 1 diabetes, and compared the results with antibody frequencies in 2425 siblings. The frequency of ICA, GADA and IA-2A was substantially higher among siblings as compared to parents of patients with type 1 diabetes (8.0% vs. 4.5%, 8.0% vs. 4.3%, and 4.5% vs. 1.9%, respectively; p<0.01). However, subdividing the probands according to age revealed a high prevalence of ICA (5.5 %), GADA (5.9 %), and IA-2A (3.1%) among parents aged 31 -40 years which was similar to that observed in siblings above 20 years of age (6.4%, 6.4%, and 3.1%). In both cohorts, GADA and IA-2A were significantly associated with the presence of ICA. The combined screening for GADA and IA-2A identified 100% of parents and 91.9% of siblings at high risk for type 1 diabetes (>10 JDF-U). Furthermore, the analysis of antibody combinations revealed that among antibody positive individuals the percentage of subjects with two or three antibodies was even higher in parents (69.0%) than in siblings (58.2%). The present study shows a high frequency of single and multiple autoantibodies in unaffected parents of patients with type 1 diabetes. Our data indicate that GAD and IA-2 not only represent the major target of autoantibodies in young siblings but also in adult relatives. These findings may be important for the design of future intervention studies.

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.