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.     

Radioimmunoassay for glutamic acid decarboxylase (GAD65) autoantibodies as a diagnostic aid for stiff-man syndrome and a correlate of susceptibility to type 1 diabetes mellitus

OBJECTIVE: To establish and validate a double-antibody radioimmunoassay (RIA) for detecting serum auto-antibodies against glutamic acid decarboxylase (GAD65). This enzyme catalyzes synthesis of the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic islet cells. MATERIAL AND METHODS: We compared the frequency of GAD65 and other "thyrogastric" autoantibodies in adult patients with stiff-man (Moersch-Woltman) syndrome, type 1 diabetes, or polyendocrine disorders and in healthy subjects. The frequency of pancreatic islet cell antibody (ICA) detection was also assessed. The GAD65 RIA was validated by testing blinded samples, by confirming the specificity of low-titered positive results by "cold" antigen inhibition, and by comparing the RIA results with results of a kit assay incorporating staphylococcal protein A as immunoprecipitant. Recombinant GAD65 protein labeled with 125I was used as antigen, and a combination of anti-human IgG and IgM was used as immunoprecipitant. Seropositivity was determined for ICA and gastric parietal cell antibodies by indirect immunofluorescence assays and for thyroid peroxidase (microsome) and thyroglobulin antibodies by agglutination assays. RESULTS: We detected GAD65-specific antibodies in all but 1 of 46 local patients with stiff-man syndrome (98%); 16 had evidence of diabetes. Positive values exceeded 20 nmol/L in 96%, and 89% were ICA-positive; 76% had additional thyrogastric antibodies. Of 41 patients with type 1 diabetes (17 local and 24 workshop serum specimens), 33 were GAD65 antibody-positive (80%); 85% of these positive values were 20 nmol/L or lower. Only 18% of sera from patients with type 1 diabetes were ICA-positive, but 59% had other thyrogastric autoantibodies. Of 20 patients with autoimmune endocrinopathies without diabetes or stiff-man syndrome, 35% were GAD65 antibody-positive, 5% were ICA-positive, and 90% were thyrogastric antibody-positive. Of 117 healthy control subjects, 8% were GAD65 antibody-positive, and a third of those had other thyrogastric antibodies (14% overall); none was ICA-positive. CONCLUSION: Seropositivity in the double-antibody RIA for GAD65 autoantibody is a sensitive and specific marker of predisposition to type 1 diabetes and related organ-specific autoimmune disorders. As such, this RIA is complemented by assays for thyroid and gastric parietal cell autoantibodies.     

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.     

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.     

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.     

Quantum dynamics of molecular motions based on a functional method

Molecular mimicry between viral antigens and host proteins was often suggested to be involved in induction of autoimmune diseases. In type 1 diabetes where pancreatic beta cells are destroyed by autoimmune phenomena, a linear sequence homology between a major autoantigen, glutamate decarboxylase (GAD), and the 2C protein of coxsackie B4 was identified. In addition, a sequence homology between GAD and the mycobacterial heat shock protein 60 was described and the suggestions were made that molecular mimicry between GAD, coxsackievirus B4-2C protein, and/or heat shock protein 60 (hsp60) may be actively involved in an autoimmune reaction towards the pancreatic beta-cells. Our group was the first to isolate human monoclonal autoantibodies to GAD (MICA 1-6) from a patient with newly diagnosed type 1 diabetes. The MICA allowed a detailed characterization of the diabetes associated self-epitopes in GAD and represent a set of GAD autoantibodies present in sera from patients with type 1 diabetes. Using deletion mutants of GAD we demonstrated that the regions of GAD covering the homology sequences to coxsackievirus B4 and to the hsp60 were absolutely required for binding of the MICA to GAD. We now designed an antibody-based analysis to ask whether molecular mimicry between GAD and coxsackie B4-2C or hsp60 is relevant in type 1 diabetes. Since part of the MICA recognize conformational epitopes, they allow to test for conformational molecular mimicry in viruses that have been incriminated in the development of type 1 diabetes. Our data reveal no crossreactivity between the diabetes associated GAD epitopes defined by the MICA and hsp60, rubellavirus, cytomegalovirus, and coxsackie B1-B6 virus antigens. Neither coxsackie B4-specific antibodies in sera from normal individuals nor GAD-positive sera from patients with type 1 diabetes indicated a crossreactivity between coxsackie B4-2C and GAD. Although the regions in GAD homologous to coxsackie B4-2C and hsp60 represented parts of GAD indispensible for binding of diabetes associated autoantibodies they did not mediate a crossreactivity of autoantibodies between GAD and these two proteins. No evidence for molecular mimicry between GAD and a whole panel of foreign antigens was detected by autoantibodies in type 1 diabetes.     

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.     

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.     

Measurements of colour constancy by using a forced-choice matching technique

In order to compare the reactivity of glutamic acid decarboxylase (GAD) antibodies recognizing linear and conformational epitopes as islet cell cytoplasmic antibodies (ICA), monoclonal antibodies were generated. An ELISA displacement test using two biotinylated monoclonals recognizing a linear (M61/7E11) or a conformational GAD65 epitope (M65/6B12) was performed to identify epitope regions recognized by monoclonal GAD antibodies. The GAD binding by monoclonal GAD antibodies was tested by immunofluorescence on fixed and unfixed pancreatic sections of human, rat, and mouse, and by Dot-blot experiments. 16/23 (69.6%) of the monoclonals were specifically reactive with GAD65 and 7/23 (30.4%) were reactive with both GAD isoforms. 8/16 (50%) of monoclonal GAD65 antibodies recognized a linear GAD epitope located at the N-terminus (pattern 1). 5/16 (31.3%) displaced M65/6B12, indicating the recognition of a conformational GAD epitope (pattern 2). Monoclonals belonging to patterns 1 and 2 showed strong ICA binding. 3/16 (18.8%) of monoclonals specific for GAD65 with weak or no immunostaining of pancreatic islets (pattern 3) did not inhibit the binding of both biotinylated antibodies in the displacement test, indicating other epitope specificities. In conclusion, GAD antibodies recognizing both conformational and linear epitopes of the GAD65 molecule are involved in ICA binding with strong reactivity. Furthermore, results obtained with monoclonals of pattern 3 suggest the occurrence of GAD65 epitopes partly inaccessible on cryosections, which may result in an ICA-negative test of GAD65 autoantibody positive sera.     

Adhesion of staphylococci to polyurethane and hydrogel-coated polyurethane catheters assayed by an improved radiolabelling technique

Glutamate decarboxylase autoantibodies (GAD65Ab) and beta-cell function were evaluated at and 3 years after diabetes onset in consecutive subjects over 15 years of age. At onset, 21/32 (66%) insulin-treated patients (mean age 43, range 16-79 years) had GAD65Ab; all GAD65Ab persisted 3 years later. At onset, 20/82 (24%) non-insulin-treated patients (mean age 56, range 20-79 years) had GAD65Ab. Of those with persistent GAD65Ab, 8 non-insulin-treated and 11 insulin-treated patients consented to follow-up glucose and glucagon stimulation tests. For non-insulin-treated patients, quantitative GAD65Ab index at onset correlated inversely with 1 + 3 min C-peptide response to glucose (r = -0.68, P < 0.05) and to glucagon (r = -0.79, P < 0.05) 3 years later. Those with high (> 0.50) initial GAD65Ab index had lower C-peptide (fasting, 1 + 3 min after glucose and after glucagon) 3 years later, versus those with low (< 0.50) initial GAD65Ab index (P < 0.05). In conclusion, not only did GAD65Ab presence predict future insulin dependence, but higher GAD65Ab levels may mark more rapid decline in beta-cell function in apparent non-insulin-dependent diabetes.     

Human B cells secreting immunoglobulin G to glutamic acid decarboxylase-65 from a nondiabetic patient with multiple autoantibodies and Graves' disease: a comparison with those present in type 1 diabetes

Antibodies to glutamic acid decarboxylase-65 (GAD65) are present in a number of autoimmune disorders, such as insulin-dependent (type 1) diabetes mellitus (IDDM), stiff man syndrome, and polyendocrine autoimmune disease. Antibodies to GAD in IDDM patients usually recognize conformation-dependent regions on GAD65 and rarely bind to the second isoform, glutamic acid decarboxylase-67 (GAD67). In contrast, those present in stiff man syndrome and polyendocrine disease commonly target the second isoform (GAD67) and include antibodies that are less dependent on the conformation of the molecule. By immortalizing peripheral blood B cells with Epstein-Barr virus, we have generated three human IgG autoantibodies, termed b35, b78, and b96, to GAD65 from one patient with multiple autoantibodies to endocrine organs and Graves' disease. All three autoantibodies are of the IgG1 isotype, with islet cell activity, and do not react with GAD67. The regions on GAD65 recognized by the three autoantibodies have been investigated by immunoprecipitation with a series of chimeras, by binding to denatured and reduced antigens, and using protein footprinting techniques. Using chimeric GAD proteins, we have shown that b35 targets the IDDM-E1 region of GAD65 (amino acids 240-435) whereas both b78 and b96 target the IDDM-E2 region of GAD65 (amino acids 451-570). Furthermore, examination of binding to recombinant GAD65 and GAD67 by Western blotting revealed some differences in epitope recognition, where only b78 bound denatured and reduced GAD65. However, b35, b78, and b96 autoantibodies had different footprinting patterns after trypsin treatment of immune complexes with GAD65, again indicating different epitope recognition. Our results indicate that antibodies to GAD65 present in nondiabetic patients with multiple autoantibodies to endocrine organs show similarities to those in IDDM (by targeting IDDM-E1 and IDDM-E2 regions of GAD65) as well as subtle differences in epitope recognition (such as binding to denatured and reduced GAD65 and by protein footprinting). Thus, the GAD65 epitopes recognized by autoantibodies in different autoimmune diseases may overlap and be more heterogeneous than previously recognized.     

Widespread expression of an autoantigen-GAD65 transgene does not tolerize non-obese diabetic mice and can exacerbate disease

Glutamic acid decarboxylase (GAD)65 is a pancreatic beta cell autoantigen implicated as a target of T cells that initiate and sustain insulin-dependent diabetes mellitus (IDDM) in humans and in non-obese diabetic (NOD) mice. In an attempt to establish immunological tolerance toward GAD65 in NOD mice, and thereby to test the importance of GAD in IDDM, we generated three lines transgenic for murine GAD65 driven by a major histocompatibility complex class I promoter. However, despite widespread transgene expression in both newborn and adult mice, T cell tolerance was not induced. Mononuclear cell infiltration of the islets (insulitis) and diabetes were at least as bad in transgenic mice as in nontransgenic NOD mice, and in mice with the highest level of GAD65 expression, disease was exacerbated. In contrast, the same transgene introduced into mouse strain, FvB, induced neither insulitis nor diabetes, and T cells were tolerant to GAD. Thus, the failure of NOD mice to develop tolerance toward GAD65 reflects at minimum a basic defect in central tolerance, not seen in animals not predisposed to IDDM. Hence, it may not be possible experimentally to induce full tolerance toward GAD65 in prediabetic individuals. Additionally, the fact that autoimmune infiltration in GAD65 transgenic NOD mice remained largely restricted to the pancreas, indicates that the organ-specificity of autoimmune disease is dictated by tissue-specific factors in addition to those directing autoantigen expression.     

Characterization of insulin autoantibodies in relatives of patients with type I diabetes

We studied in detail the anti-insulin autoantibodies in 29 nondiabetic relatives of patients with type I diabetes. The affinity of the autoantibodies for [125I]human insulin was high (1.34 x 10(9)-20.71 x 10(9) L/mol), and the capacity was low (0.84 x 10(-12)-37.80 x 10(-12) M). The product of affinity x capacity of each relative's antibodies directly correlated (r = 0.99) with the level of antibodies determined in our standard radioassay. The autoantibodies from each of the subjects studied had the same rank order of affinities for insulin from different species. Guinea pig, fish insulin, and insulin containing Trp rather than Leu in position 13 of the A-chain inhibited minimally the human insulin binding. Human proinsulin, insulin containing Gln rather than Glu in position 17 of the A-chain, and desoctapeptide insulin (des B23-30) all inhibited binding effectively. Insulin autoantibodies in relatives of patients with type I diabetes share common epitope(s), which suggests a common pathogenic mechanism for production of such antibodies. The epitopes from this initial analysis appear to include amino acids B1-B3 and A8-A13. The region recognized can be distinguished from the insulin receptor binding domain.     

Severe complications of reflex sympathetic dystrophy: infection, ulcers, chronic edema, dystonia, and myoclonus

To study the relationship of IA-2 antibodies (IA-2A) to other autoantibodies and genetic risk markers in insulin-dependent diabetes mellitus (IDDM), 758 children and adolescents younger than 15 years of age (mean age 8.4 years) with newly diagnosed diabetes were analysed for IA-2A, GAD antibodies (GADA) and insulin autoantibodies (IAA) with radiobinding assays, for islet cell antibodies (ICA) with immunofluorescence and for HLA DR alleles by serology. IA-2A were detected in 85.9% of cases with no association with gender or age. An overwhelming majority of the patients (71.3%) tested positive for three or more antibodies, and 90.7% for at least two. Fifty-four subjects (7.1%) had one antibody detectable, whereas only 2.1% of the patients tested negative for all four. A higher proportion of patients was positive for IA-2A and/or GADA than for ICA alone (95.5 vs 84.2%, p < 0.001). The prevalence and level of IA-2A were increased in cases carrying HLA DR4/non-DR3 compared with other DR combinations. The results indicate that almost all patients with newly diagnosed childhood IDDM can be identified by screening with these four autoantibodies. The combination of IA-2A and/or GADA had a higher sensitivity for IDDM than ICA alone. The close association between IA-2A and HLA DR4, the strongest single allele predisposing to IDDM, suggests that IA-2A may be a more specific marker of beta-cell destruction than GADA, which have been shown to associate with the DR3 allele and thyroid autoimmunity.     

GAD antibodies in NIDDM. Ten-year follow-up from the diagnosis

OBJECTIVE: To study the frequency of antibodies to glutamic acid decarboxylase (GAD) and islet cell antibodies (ICAs) and their predictive value with respect to the development of insulin deficiency in 133 newly diagnosed middle-aged patients with non-insulin-dependent diabetes mellitus (NIDDM) and in 126 control subjects and to study the persistence of GAD antibodies in diabetic patients during the follow-up. RESEARCH DESIGN AND METHODS: The study participants consisted of a well-characterized group of 133 middle-aged newly diagnosed patients with NIDDM and 126 control subjects. The follow-up examinations were performed 5 and 10 years after the baseline. The development of absolute and relative insulin deficiency was based on a stimulated C-peptide level that was undetectable or < 0.70 nmol/l, respectively. GAD antibodies were measured retrospectively from stored samples. RESULTS: The overall prevalence of GAD antibody and ICA positivity at the time of diagnosis was 9.0 and 3.8% in diabetic patients and 1.6 and 0% in the control population, respectively. During the 10-year follow-up, 3 (2.3%) and 10 (7.5%) of the diabetic patients developed absolute and relative insulin deficiency, respectively. Of these, two (67%) and six (60%) had been GAD antibody-positive at the time of diagnosis. The sensitivity and specificity of the GAD antibody to predict absolute or relative insulin deficiency were 67 vs. 94% and 60 vs. 95%, while corresponding figures for ICA were 33 vs. 97% and 20 vs. 98%, respectively. The negative predictive value of GAD antibody testing was higher than positive predictive value (97 vs. 50%). During the follow-up, low-grade GAD antibody positivity showed an evanescent nature, whereas the high levels were quite persistent. CONCLUSIONS: In an unselected population of newly diagnosed NIDDM patients, the prevalence of latent autoimmune diabetes in adults was < 10%. While GAD antibody and ICA measured at the time of diagnosis of NIDDM are equally specific predictors of subsequent insulin dependency, the GAD antibody may have a higher sensitivity. Therefore, measurements of GAD antibody may aid the clinician in the choice of treatment of these patients.     

Effect of oral and intravenous insulin and glutamic acid decarboxylase in NOD mice

Islet cell antigens have been administered orally and intravenously (I.V.) to NOD mice to assess their abilities to protect from or delay the onset of diabetes, and thereby provide insights that may have therapeutic implications in human trials. Whereas we and others have observed a delay in the onset of diabetes in NOD mice that have been fed with insulin from early life, we report here for the first time that feedings with porcine GAD65 alone (p = 0.226) or in combination with insulin (p = 0.011), have anti-diabetic effects in a prolonged study period (>400 days). While antigen-specific inhibitions of in vitro lymphocytic proliferation responses were seen (p < 0.05), antibody levels were unaffected by oral antigen treatments. IFN-gamma mRNA levels were downregulated in the islet infiltrates following oral antigen treatments while IL-2 and TNF-beta were expressed in all instances. We also observed that I.V. human recombinant GAD65, and porcine GAD given at weaning, delayed diabetes onset (p = 0.004) while similar treatments with a variety of inactive insulin preparations were generally ineffective. These findings thus indicate varying effects of oral and I.V. autoantigen administrations on the development of diabetes in NOD mice, and describe the immunological processes induced by oral autoantigen treatments.     

Metabolic and immune parameters at clinical onset of insulin-dependent diabetes: a population-based study. IMDIAB Study Group. Immunotherapy Diabetes

The age at diagnosis of insulin-dependent diabetes mellitus (type I DM) varies between childhood and adulthood. The aim of this study was to define the immunologic and metabolic characteristics of the disease according to the age at which it is diagnosed. We evaluated the residual beta-cell function (basal and stimulated C-peptide) and frequency of two major islet cell-related autoantibodies, glutamic acid decarboxylase (GAD) and tyrosine phosphatase-like molecule (IA-2ic), at the onset of type I DM. A population-based study was performed with 235 consecutive cases of recent-onset (<4 weeks) type I DM (ages 5 to 45 years) diagnosed in the Lazio region of central Italy. Five age groups were considered: patients diagnosed between ages 5 and 7 years (n = 10), 7 and 10 years (n = 38), 10 and 17 years (n = 94), 17 and 20 years (n = 17), and 20 and 45 years (n = 76). Patients diagnosed before puberty had significantly reduced C-peptide secretion compared with patients diagnosed at a later age (P < .02). Glycosylated hemoglobin (HbA1c) did not differ at diagnosis between the different age groups. Patients diagnosed at puberty or after required significantly less insulin compared with younger patients (P < .04). GAD antibodies were found in 65% and IA-2ic antibodies in 59% of patients. GAD antibodies tended to be more frequent in patients diagnosed after age 17 compared with younger patients (P = .05), while IA-2ic antibodies were not age-related. These data suggest that (1) the extent of beta-cell damage differs between patients diagnosed before and after puberty, the process being more destructive in children less than 7 years of age, when C-peptide levels are the lowest; and (2) residual beta-cell function at diagnosis is not influenced by the presence or absence of islet cell-related antibodies. These findings have implications for trials in type I DM diagnosis aimed at protecting beta cells from end-stage destruction and in attempts to prevent the disease in susceptible individuals.     

Population based study of prevalence of islet cell autoantibodies in monozygotic and dizygotic Danish twin pairs with insulin dependent diabetes mellitus

OBJECTIVE: To study the comparative importance of environment and genes in the development of islet cell autoimmunity associated with insulin dependent diabetes mellitus. DESIGN: Population based study of diabetic twins. SETTING: Danish population. SUBJECTS: 18 monozygotic and 36 dizygotic twin pairs with one or both partners having insulin dependent diabetes. MAIN OUTCOME MEASURES: Presence of islet cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase (GAD65) in serum samples from twin pairs 10 years (range 0-30 years) and 9.5 years (2-30 years) after onset of disease. RESULTS: In those with diabetes the prevalence of islet cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase in the 26 monozygotic twins was 38%, 85%, and 92%, respectively, and in the dizygotic twins was 57%, 70%, and 57%, respectively. In those without diabetes the proportions were 20%, 50%, and 40% in the 10 monozygotic twins and 26%, 49%, and 40% in the 35 dizygotic twins. CONCLUSION: There is no difference between the prevalence of islet cell autoantibodies in dizygotic and monozygotic twins without diabetes, suggesting that islet cell autoimmunity is environmentally rather than genetically determined. Furthermore, the prevalence of islet cell antibodies was higher in the non-diabetic twins than in other first degree relatives of patients with insulin dependent diabetes. This implies that the prenatal or early postnatal period during which twins are exposed to the same environment, in contrast with that experienced by first degree relatives, is of aetiological importance.     

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.