Recognition of glutamic acid decarboxylase (GAD) by autoantibodies from different GAD antibody-positive phenotypes

Autoantibodies against the smaller isoform of glutamic acid decarboxylase (GAD) are markers for Type 1 diabetes. GAD65 autoantibody (GAD65Ab)-positive individuals in the general population are, however, mostly at low risk of developing Type 1 diabetes, suggesting that GAD65Ab phenotypes may be associated with different underlying pathogenic processes. The aim of this study was to test the hypothesis that Type 1 diabetes patients (n = 243; group I), GAD65Ab-positive healthy individuals (n = 28; group II), and healthy first-degree relatives of Type 1 diabetes patients (n = 41; group III) have antibody phenotypes that recognize different GAD65 epitopes. Sera from groups I-III were tested for their binding to GAD65 and GAD67, as well as six different GAD65/67 fusion proteins. Regardless of group, sera reactive to both GAD65 and GAD67 showed broader epitope reactivity than GAD65-specific sera. Furthermore, Type 1 diabetes patients showed a more restricted epitope binding than healthy individuals and first-degree relatives, demonstrating significantly less binding to the N-terminal part of GAD65 and to GAD67. Our analysis demonstrates that the N-terminal part is essential for full antibody binding to GAD65, in particular, to the middle epitope. It is suggested that Type 1 diabetes is associated with restricted GAD65Ab epitope specificity.     

GAD65 autoantibody epitopes in adult patients with latent autoimmune diabetes following GAD65 vaccination.

AIMS: Subcutaneous injection of recombinant human GAD65 (rhGAD65) in patients with latent autoimmune diabetes in adults (LADA) correlates with an increase in C-peptide levels. In this study we analysed the effect of rhGAD65 administration on the GAD65-specific autoimmune response. METHODS: Longitudinal serum samples obtained from LADA patients (n = 47) who received 4, 20, 100 or 500 microg alum-formulated rhGAD65 or placebo by subcutaneous injection twice (4 weeks apart) were analysed for their epitope recognition using GAD65-specific recombinant Fab and GAD65/67 fusion proteins. RESULTS: Overall, minor changes in the epitope pattern were observed using either approach. Only in the 500-microg dosage group was an increase in GAD65Ab level associated with a significant increase in the binding to a conformational epitope located at the middle part of GAD65. CONCLUSIONS: Our data suggest that the apparent beneficial effects of 20 microg alum-formulated recombinant human GAD65 is not explained by changes in the GAD65Ab epitope pattern.     

Autoreactive epitopes defined by diabetes-associated human monoclonal antibodies are localized in the middle and C-terminal domains of the smaller form of glutamate decarboxylase.

The gamma-aminobutyrate-synthesizing enzyme glutamate decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) is a major target of autoantibodies associated with both early and late stages of pancreatic beta-cell destruction and development of type 1 diabetes. We have used five monoclonal anti-islet-cell antibodies (MICAs 1,2,3,4, and 6) derived from a newly diagnosed diabetic patient to probe the autoimmune epitopes in the enzyme. All the MICAs specifically recognized the smaller GAD protein, GAD65, and did not recognize the nonallelic GAD67 protein. A series of N-terminal, C-terminal, and internal deletion mutants, as well as protein footprinting, were used to identify the target regions in GAD65. Immunoprecipitation revealed two major native epitope areas in the GAD65 molecule. The first, defined by MICAs 1 and 3, is destroyed by deleting 41 amino acids at the C terminus but is also dependent on intact amino acids 244-295. This epitope (or epitopes) may span both middle and C-terminal domains of the protein. The second conformational epitope region, defined by MICAs 4 and 6, is dependent on intact amino acids 245-295 but is not affected by deletion of 110 amino acids at the C terminus and is therefore confined to domain(s) in the middle of the molecule. MICA 2 recognizes a linear epitope close to the C terminus. Thus, the N-terminal domain of GAD65, which differs most significantly from GAD67, does not harbor the MICA epitopes. Rather subtle amino acid differences in the middle and C-terminal domains define the GAD65-specific autoimmune epitopes. Analysis of sera from 10 type 1 diabetic patients suggests that MICAs 1, 3, 4, and 6 represent a common epitope recognition in this disease, whereas the MICA 2 epitope is rare. Furthermore, autoantibodies in some sera are restricted to the MICA 1/3 epitope, suggesting that this epitope may represent a single dominant epitope in the early phases of beta-cell autoimmunity.     

Murine monoclonal glutamic acid decarboxylase (GAD)65 antibodies recognize autoimmune-associated GAD epitope regions targeted in patients with type 1 diabetes mellitus and Stiff-man syndrome

To study the immune response to glutamic acid decarboxylase (GAD) in insulin-dependent diabetes mellitus, monoclonal GAD antibodies after fusion of splenocytes from a nondiabetes-susceptible BALB/c mouse immunized with human recombinant GAD65 were generated. Of the 44 monoclonals, 35 are specific for the GAD65 isoform, whereas 9 also react with GAD67. Some 37 monoclonals, including all GAD65/67 reactive antibodies, react with GAD by Western blot analysis. The remaining 7 GAD65 monoclonals bind GAD only in an immunoprecipitation assay, which implies that they target epitopes dependent on the conformation of the GAD molecule. The¹²⁵I-GAD binding of the GAD65 monoclonals reactive on Western blotting was significantly diminished by all 3 sera from Stiff-man syndrome patients but only by 3/30 (10%) sera from type 1 diabetic patients. In contrast, the 7 monoclonal antibodies reactive with a conformation-dependent GAD epitope were competitive with 83% of GAD-autoantibody-positive sera from these diabetic patients. Using chimeric GAD65/67 proteins, the epitope region targeted by these monoclonals was mapped to the middle of GAD65 (amino acids 221–442). This central conformation-dependent GAD region was also targeted by sera from patients with type 1 diabetes. In conclusion, our data show that evne after common immunization of a nondiabetes-susceptible mouse strain, monoclonals were obtained which preferentially react with the GAD65 linear amino-terminus (amino acids 4–17) and a conformation-dependent region located in the middle of GAD targeted by autoantibodies, indicating that this GAD region is not restricted to the autoimmune response associated with the Stiff-man syndrome and the bete-cell destruction in type 1 diabetes mellitus.     

Unique epitopes of glutamic acid decarboxylase autoantibodies in slowly progressive type 1 diabetes

Disease-specific epitope profiles of glutamic acid decarboxylase (GAD)65 autoantibodies (GAD65Ab) were studied in slowly progressive type 1 (insulin-dependent) diabetes mellitus (SPIDDM) and acute onset type 1 (insulin-dependent) diabetes mellitus (AIDDM) using seven kinds of GAD65/67 chimeric molecules. Sera obtained from Japanese SPIDDM (n = 17) and AIDDM (n = 46) patients followed prospectively were analyzed by immunoprecipitation, ELISA, and Western blotting. GAD65Ab in all SPIDDM samples reacted specifically with an N-terminal linear epitope located on the membrane anchoring domain between amino acids 17-51 and C-terminal conformational epitope between amino acids 443-585 of GAD65. The binding of GAD65Ab with N-terminal 83 residues in SPIDDM inversely correlated with the period in which insulin was not required. GAD65Ab in AIDDM did not react with N-terminal epitope located between amino acids 1-83, irrespective of the titer of GAD65Ab. A novel epitope of GAD65Ab in AIDDM residing between amino acids 244-360 was identified in 17% (8 of 46) of patients whose age of onset was younger than other AIDDM patients. In conclusion, GADAb in SPIDDM has unique N-terminal linear epitopes that are located on the anchoring domain of GAD65 molecules. Association is suggested between GAD65Ab targeted to this region and slowly progressive beta-cell failure in SPIDDM.     

Association of amino-terminal-specific antiglutamate decarboxylase (GAD65) autoantibodies with beta-cell functional reserve and a milder clinical phenotype in patients with GAD65 antibodies and ketosis-prone diabetes mellitus

CONTEXT: We previously characterized patients presenting with diabetic ketoacidosis prospectively into four subgroups of ketosis-prone diabetes mellitus (KPDM), based on the presence or absence of beta-cell autoimmunity (A+ or A-) and beta-cell functional reserve (B+ or B-). The A+B- KPDM subgroup comprises patients with classic, autoimmune type 1 diabetes, whereas the A+B+ KPDM subgroup has only partial beta-cell loss and a distinct clinical phenotype. OBJECTIVE: We hypothesized that epitope specificity of autoantibodies directed against the 65-kDa isoform of glutamate decarboxylase (GAD65) reflects differences in beta-cell destruction. DESIGN: Sera of sequential GAD65Ab-positive KPDM patients admitted for diabetic ketoacidosis (n = 36) were analyzed for their epitope recognition using five GAD65-specific recombinant Fab and their ability to inhibit GAD65 enzymatic activity. All patients were followed longitudinally to assess beta-cell functional reserve and insulin dependence. RESULTS: Binding to an amino-terminal epitope defined by monoclonal antibody DPD correlated positively with fasting serum C-peptide levels at baseline (P = 0.0008) and after 1 yr (P = 0.007). Binding to the DPD-defined epitope also correlated positively with area under the curve for C-peptide after glucagon stimulation (P = 0.007) and with homeostasis model assessment percent B at 1 yr (P = 0.03). Binding to the DPD-defined epitope was significantly stronger in A+B+ than in A+B- patients (P = 0.001). Sera of 16 patients (44%) significantly inhibited GAD65 enzymatic activity, but this did not correlate with beta-cell function. CONCLUSION: DPD-defined epitope specificity is correlated directly with preserved beta-cell functional reserve in GAD65Ab-positive patients and is associated with the milder clinical phenotype of A+B+ KPDM.     

GAD65-Specific Cytotoxic T Lymphocytes Mediate Beta-Cell Death and Loss of Function

Autoimmunity to islet cell antigens like glutamic acid decarboxylase 65kD (GAD65) is associated with the destruction of insulin-producing β-cells and progression to type 1 diabetes (T1D) in NOD mice and humans. T cell responses to GAD65 are detectable in the spleen of prediabetic NOD mice and in the peripheral blood of humans prior to the onset of overt hyperglycemia. Previous findings from our lab revealed that GAD65_546-554-specific cytotoxic T lymphocytes (CTL) are present in naïve NOD mice and are able to induce islet inflammation upon adoptive transfer into NOD.scid recipients. Additionally, we found that professional antigen-presenting cells (APC) generate the p546-554 epitope from a soluble GAD65 fragment, p530-554, and from GAD65 released by injured β-cells in vivo. Here, we report that the GAD65 fragment p546-554 is a dominant CTL-inducing epitope which is naturally processed and presented by a GAD65-expressing β-cell line. Further, co-culture of GAD65_546-554-specific CTL with the β-cells leads to a reduction in insulin production and the induction of perforin-mediated cell death. Collectively, these findings support a role for the cross-presentation of GAD65 antigen in the priming and enhancement of dominant GAD65-specific CTL responses, which can directly target β-cells that display GAD65 epitopes.     

GAD65 antibody prevalence and association with thyroid antibodies, HLA-DR in Chinese children with type 1 diabetes mellitus

Persistent humoral autoimmunity to the enzyme glutamic acid decarboxylase (GAD) has been described in a substantial proportion of patients with type 1 diabetes mellitus. Higher prevalence of GAD antibody in diabetes patients using a new radioligand-binding assay with recombinant human GAD65 antibodies (GAD65Ab) has been seen in several studies. Using this method, we have reassessed the prevalence of GAD65Ab and investigated the association of GAD65Ab with HbA1C values, C-peptide values, HLA-DR typing and thyroid autoimmune antibody in 70 Chinese children with type 1 diabetes mellitus (mean age of onset 8.21+/-3.84 years, mean duration 3.39+/-2.54 years). Our result revealed that GAD65 antibodies were present in 54.3% (38/70) of diabetes children. There was no significant difference in gender, diabetes onset and duration, HbA1c, C-peptide concentration and frequencies of HLA DR3, DR4, DR9, DR3/DR4, DR3/DR9 and DR4/DR9 genotypes between GAD65Ab+ and GAD65Ab- groups. There was no negative correlation between GAD65Ab values and duration of diabetes in those with GAD65Ab positivity (r=-0.239, P>0.05). The frequencies of antimicrosomal and anti-thyroglobulin antibodies in GAD65Ab+ (13.5,8.1%, respectively) were not different from GAD65- patients (9.4,12.5%, respectively).     

Diagnostic sensitivity of immunodominant epitopes of glutamic acid decarboxylase (GAD65) autoantibodies in childhood IDDM

Summary The prevalence and titre of epitope-specific autoantibodies to glutamic acid decarboxylase (GAD65) in 155 insulin-dependent diabetic (IDDM) and 9 GAD65 antibody (Ab)-positive healthy children were determined using four GAD65/67 chimaeric molecules which discriminate among the N-terminal (N), middle (M) and C-terminal (C) epitopes of GAD65. Radioligand binding assays for IgG Ab used immunoprecipitation of in vitro translated ³⁵S-GAD. We found autoantibodies to GAD65 in 116 of 155 (75%), to GAD67 in 19 of 155 (12%) (p<0.0001) and to the GAD65-N-67 chimaera in 25 of 155 (16%) (p<0.0001) IDDM sera. GAD67Ab were found almost exclusively (17 of 19, 89%) in GAD65Ab-positive sera and the levels of GAD67Ab correlated with those of GAD65Ab (r ²=0.5913; p=0.009). GAD65Ab directed to GAD65-M were found in 104 of 155 (67%), to GAD65-C in 104 of 155 (67%) and to GAD65-M + C in 116 of 155 (75%) of IDDM sera, and indicated reactivity to at least two distinct epitopes. Among the nine GAD65Ab-positive healthy children, two (22%) were also positive with GAD67, nine (100%) with GAD65-M + C, seven (78%) with GAD65-M, eight (89%) with GAD65-C and two (22%) with GAD65-N-67. Titres of GAD65Ab (p=0.007), GAD65-C-Ab (p=0.002) and GAD65C + M-Ab (p=0.003), but not of GAD65-M-Ab (p=0.101) were significantly higher in IDDM than in healthy children. We conclude that GAD65Ab in IDDM and healthy children are directed to middle and C-terminal epitopes, and propose that levels of antibodies specifically directed to the carboxy-terminal end of GAD65 may distinguish IDDM from healthy children.Abbreviations Ab Antibodies - GAD glutamic acid decarboxylase - ICA islet cell antibodies - IDDM insulin-dependent diabetes mellitus - JDF Juvenile Diabetes Foundation - PC-2 proinsulin-converting enzyme 2 - TCA trichloroacetic acid - M middle terminal epitope - N N-terminal epitope - C C-terminal epitope     

Autoantibody recognition of COOH-terminal epitopes of GAD65 marks the risk for insulin requirement in adult-onset diabetes mellitus

Some type 2 diabetic subjects develop secondary failure to sulphonylurea treatment and require insulin therapy. To test the diagnostic sensitivity and specificity of epitopes of GAD65 autoantibodies (GAD65Ab) for insulin requirement, in patients with latent autoimmune diabetes of the adult, we studied 569 adult subjects with a clinical diagnosis of type 2 diabetes mellitus. All the patients had been initially treated with hypoglycemic agents and/or diet for at least 1 yr. The presence of GAD65Ab (61/569, 10.7%) depended on insulin therapy (P<0.0001), low BMI (P<0.0001), and low basal C-peptide (P = 0.01). The majority of GAD65Ab-positive subjects (47/61, 77%) had antibodies directed to both middle (GAD65-MAb) and COOH-terminal (GAD65-CAb) epitopes. However, GAD65-CAb were more frequent in insulin-treated subjects (92% of GAD65Ab+ individuals) than in subjects treated with hypoglycemic agents and/or diet (18.2% of GAD65Ab+ individuals), while the exclusive presence of GAD65-MAb was more frequent in subjects treated with hypoglycemic agents and/or diet (81.8% vs. 8%) (P<0.0001). The presence of GAD65-CAb had a diagnostic specificity for insulin requirement as high as 99.4% (compared with 96.9% of GAD65Ab as measured in the traditional radiobinding assay) and identified a subgroup of patients with low BMI, low basal C-peptide values, and a need for insulin therapy. Subjects carrying only GAD65-MAb were phenotypically indistinguishable from GAD65Ab-negative patients. Patients positive for GAD65-M+CAb, but not those positive for GAD65-MAb only, showed an increased risk for thyroid autoimmunity, as revealed by the presence of thyroid peroxidase autoantibodies. Our study demonstrates that the use of epitope-specific antibody assays improves the diagnostic specificity of GAD65Ab, and that the presence of GAD65Ab binding to COOH-terminal epitopes is strongly associated with a need for insulin requirement.     

Epitope spreading and a varying but not disease-specific GAD65 antibody response in Type I diabetes

Aims/hypothesis. The aim of this study was to analyse the conformational and linear epitope profiles of glutamic acid decarboxylase antibody (GAD65-ab)-positive sera to find disease-specific epitope profiles and to study, whether GAD65-ab epitope recognition changes or spreads during the prediabetic period and, thus, can provide markers to differentiate early from later stages of progression to diabetes.¶Methods. Sera from subjects before (n = 21), at onset (n = 44), or at increased risk of Type I (insulin-dependent) diabetes mellitus (n = 20) and from patients with stiff-man syndrome (SMS, n = 18) or polyendocrine autoimmune syndrome (PAS, n = 21) were analysed for conformational and linear GAD65 epitope recognition by an immunohistochemical blocking test based on human monoclonal GAD65-ab (MICA 1–10) and western blotting of a GAD65 epitope-cDNA-library.¶Results. A redundant reactivity of many GAD65-ab positive sera to three major conformational (EP-1, EP-2, EP-3) and two dominant linear epitope clusters (amino acid 1–124 and 535–585) was observed in diabetes, polyendocrine autoimmune syndrome and stiff-man syndrome and no disease-specific epitopes or epitope-profiles were detected. Epitope recognition broadened with higher titres and with the vulnerability of patients to acquire additional autoimmune diseases apart from diabetes. Low GAD65-ab serum titres ( < 1200 arbitrary units) and EP-1 recognition in the absence of EP-2 binding characterised the early immune response. Changing epitope profiles combined stable recognition of EP-1 with gain or loss of reactivity to C-terminal epitopes during follow-up.¶Conclusion/interpretation. A maturing autoantibody response, which could spread from EP-1-recognition to other regions of GAD65, resulted in titre-related rather than disease-specific epitope profiles which were not sufficient to predict whether GAD65-ab positive subjects will progress to Type I diabetes, autoimmune polyendocrine syndrome or stiff-man syndrome. [Diabetologia (2000) 43: 210–217]     

Early epitope- and isotype-specific humoral immune responses to GAD65 in young children with genetic susceptibility to type 1 diabetes

OBJECTIVE: The pattern of the humoral immunity to disease-associated autoantigens may reflect the severity of the autoimmune disease process. The purpose of this study was to delineate the maturation of the humoral immunity to one of the main autoantigens in type 1 diabetes (T1D), glutamic acid decarboxylase (GAD65). DESIGN AND METHODS: Serum samples were obtained for the detection of epitope- and isotype-specific antibodies sequentially with short intervals from 36 young children with HLA-conferred genetic susceptibility to T1D starting from the first appearance of GAD65Ab. During prospective observation, ten children developed T1D. Antibodies were analyzed using biotinylated anti-human immunoglobulin (Ig) antibodies and chimeric GAD molecules in radio-binding assays. RESULTS: The immune response to GAD65 started as reactivity to the middle region and spread rapidly to the C-terminal region. IgG1 antibodies dominated among the isotypes from the first appearance of GAD65Ab, while other IgG subclasses were observed to a lesser extent. IgG4 antibodies emerged clearly as the last IgG subclass. A broad initial response comprising three to four IgG subclasses and the lack of an emerging IgG4 response during follow-up was associated with increased risk for progression to clinical diabetes (P<0.05). CONCLUSIONS: The humoral response to GAD65 epitope clusters is relatively uniform in young children, whereas there is conspicuous individual variation in IgG subclass responses except for IgG1. A narrow initial IgG subclass response to GAD65 and the emergence of IgG4 antibodies were characteristic of those who remained non-diabetic over the first few years of GAD65 autoimmunity.     

Prevention of type I diabetes transfer by glutamic acid decarboxylase 65 peptide 206-220-specific T cells

Glutamic acid decarboxylase (GAD) 65 is one of the major pancreatic antigens targeted by self-reactive T cells in type I diabetes mellitus. T cells specific for GAD65 are among the first to enter inflamed islets and may be important for the initiation of autoimmune diabetes. However, we previously reported that nonobese diabetic (NOD) mice transgenic for a T cell antigen receptor (TCR) specific for one of the immunodominant epitopes of GAD65, peptide 286-300 (G286), are protected from insulitis and diabetes. To examine whether other GAD65-reactive T cells share this phenotype, we have generated TCR transgenic NOD mice for a second immunodominant epitope of GAD65, peptide 206-220 (G206). As in G286 mice, G206 mice do not develop islet inflammation or diabetes. When adoptively transferred along with diabetogenic T cells, activated G206 T cells significantly delayed the onset of diabetes in NOD.scid recipients. Both G206 and G286 T cells produce immunoregulatory cytokines IFN-gamma and IL-10 at low levels when activated by cognate antigens. These data suggest that GAD65-specific T cells may play a protective role in diabetes pathogenesis by regulating pathogenic T cell responses. A better understanding of the functions of autoreactive T cells in type I diabetes will be necessary for choosing desirable targets for immunotherapy.     

The lack of anti-idiotypic antibodies, not the presence of the corresponding autoantibodies to glutamate decarboxylase, defines type 1 diabetes

Autoantibodies to glutamate decarboxylase 65 (GAD65Ab) are commonly believed to be a major characteristic for type 1 diabetes (T1D). We investigated the presence of GAD65Ab in healthy individuals (n = 238) and first-degree relatives (FDRs) of T1D patients (n = 27) who tested negative for GAD65Ab in conventional RIAs. Sera were applied to affinity columns coated with GAD65-specific mAbs to absorb anti-idiotypic antibodies (anti-Ids). The absorbed sera were analyzed for binding to GAD65 by RIAs. Both healthy individuals and FDRs present GAD65Ab that are inhibited by anti-Id, masking them in conventional detection methods. The presence of GAD65Ab-specific anti-Ids was confirmed by competitive ELISA. Remarkably, T1D patients (n = 54) and Stiff Person Syndrome patients (n = 8) show a specific lack of anti-Ids to disease-associated GAD65Ab epitopes. Purified anti-Ids from healthy individuals and FDRs inhibited the binding of GAD65Ab from T1D patients to GAD65. We conclude that masked GAD65Ab are present in the healthy population and that a lack of particular anti-Ids, rather than GAD65Ab per se, is a characteristic of T1D. The lack of these inhibitory antibodies may contribute to T cell activation by GAD65Ab.     

Identification and modulation of a naturally processed T cell epitope from the diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65)

T cell recognition of autoantigens is critical to progressive immune-mediated destruction of islet cells, which leads to autoimmune diabetes. We identified a naturally presented autoantigen from the human islet antigen glutamic acid decarboxylase, 65-kDa isoform (GAD65), by using a combination of chromatography and mass spectrometry of peptides bound by the type I diabetes (insulin-dependent diabetes mellitus, IDDM)-associated HLA-DR4 molecule. Peptides encompassing this epitope-stimulated GAD65-specific T cells from diabetic patients and a DR4-positive individual at high risk for developing IDDM. T cell responses were antagonized by altered peptide ligands containing single amino acid modifications. This direct identification and manipulation of GAD65 epitope recognition provides an approach toward dissection of the complex CD4(+) T cell response in IDDM.     

Autoantibodies and human leucocyte antigen class II in first-degree family members of Mexican-American type 1 diabetic patients

As part of a genetic study of type 1 diabetes in Mexican-Americans, 360 first-degree relatives of 108 type 1 diabetic probands were studied. Islet cell antibody (ICA), insulin autoantibody, glutamic acid decarboxylase (GAD(65)), and protein tyrosine phosphatase autoantibodies were measured and human leucocyte antigen (HLA) class II alleles DRB1 and DQB1 genotyping was performed. ICA was positive in 37% of the probands and 5.8% of the relatives. A subgroup of 26 probands (12 ICA+, 14 ICA-) was tested for GAD(65) and was found positive. 4/14 ICA+ first-degree relatives were GAD(65) positive. Four relatives, positive for two antibodies, subsequently developed type 1 diabetes. Life-Table analysis of first-degree relatives with autoantibodies indicated an 80% disease-free survival at 3.5 yr. HLA-DRB1 was found to be associated with the presence of ICA in both probands and relatives, whereas HLA-DPB1 was associated with autoantibody in relatives of type 1 diabetic probands. These results suggest that autoimmunity occurs in type 1 diabetes families of Mexican descent in similar frequencies to that of non-Hispanic, Caucasian families. The presence of autoantibodies appears to be regulated in part by HLA class II genes, even in the absence of overt diabetes.     

The Development and Application of HLA Tetramers in the Detection, Characterization and Therapy of Type 1 Diabetes Mellitus

Islet antigens are presented by human leukocyte antigen (HLA) class I and II molecules and are recognized by CD8(+) and CD4(+) autoreactive T cells in type 1 diabetic individuals. Early identification of individuals at risk for the disease by detection of these antigens and the autoreactive cells themselves is essential for understanding pathogenesis and for intervention at an early stage to prevent ongoing beta-cell destruction. However, the methods of identifying autoimmune development at an early stage have appeared to be limited because of the heterogeneity of the disease. The appearance of autoantibodies in preclinical type 1 diabetes mellitus (T1DM) does not follow specific patterns and depends on patient characteristics such as age. Also, results obtained with cytokine assays revealed that the number of islet antigen-responsive T cells present in the pool of peripheral blood mononuclear cells (PBMC) of non-diabetic individuals is highly variable and can be similar to that assayed in diabetics. Therefore, new identification and detection methods are needed. In this context, the use of HLA epitopes to generate stable HLA epitope tetramers has recently proved to be a promising approach to the detection of autoreactive T cells in antigen-stimulated PBMC cultures from diabetic and pre-diabetic subjects. HLA class II tetramers have been found to be capable not only of detecting TCRalphabeta of different avidities for a common ligand, e.g. GAD65(555-567(mimitope)), but also of inducing apoptosis in lymphocytes with high TCRalphabeta avidity for this ligand. This observation even opens up a potential application of HLA class II tetramers as therapeutic agents for immune intervention in T1DM.     

Characterization of the humoral immune response to glutamic acid decarboxylase in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and/or type 1 diabetes

OBJECTIVE: A humoral autoimmune response to glutamic acid decarboxylase (GAD65) is common both in patients with type 1 diabetes and in those with the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, while overt type 1 diabetes is relatively rarely diagnosed in APECED patients. The aim of this study was to assess whether this difference in the incidence of type 1 diabetes is associated with variability in the humoral immune response to GAD65, one of the major autoantigens in type 1 diabetes. METHODS: Epitope- and isotype-specific GAD65 autoantibodies were analysed in 20 patients with APECED and 20 patients with newly diagnosed type 1 diabetes alone by radiobinding assays. RESULTS: GAD65 autoantibodies targeted the middle and carboxy-terminal regions of GAD65 and occasionally the amino-terminal region in the APECED patients and comprised mainly the IgG1 subclass and less frequently the IgG2 and IgG4 subclasses. The profile of epitope- and isotype-specific GAD65 autoantibodies was similar in type 1 diabetes and APECED, except that IgG2 subclass antibodies were observed more often and at higher levels in the patients with type 1 diabetes alone (P < 0.05). None of the measured parameters separated APECED patients with type 1 diabetes from those without type 1 diabetes. CONCLUSION: APECED-associated humoral autoimmunity to GAD65 does not differ markedly from that observed in type 1 diabetes; only IgG2-GAD65 antibodies may be more closely associated with the latter entity.     

Species-specific autoantibodies in type 1 diabetes

GAD65 autoantibodies (GAD65Ab) are important markers for type 1 (insulin-dependent) diabetes mellitus. Although most patients have GAD65Ab at the time of clinical diagnosis, there are also GAD65Ab-positive individuals in the population at low risk of developing type 1 diabetes. The aim of this study was to test the hypothesis that the GAD65Ab reactivity to GAD65 cloned from human, mouse, and rat in newly diagnosed type 1 diabetic patients differ from antibody-positive healthy individuals. Sera from 254 new-onset 0- to 34-yr-old type 1 diabetic patients and 270 controls were assayed for their reactivity to human, mouse, and rat GAD65. Among the type 1 diabetic patients there was a significant better binding of human GAD65 compared to either mouse (P = 0.03) or rat GAD65 (P = 0.0005). The preference for human GAD65 increased with increasing age at onset (P = 0.0002). This differentiation was not observed in 88 GAD65Ab-positive control subjects. Our data indicate that recognition of epitopes by GAD65Ab in type 1 diabetes is different from that in nontype 1 diabetes, GAD65Ab-positive individuals.     

The predictive value of diabetes-related antibodies in children with type 1 diabetes mellitus and their siblings

The purpose of this study is to screen for diabetes-related antibodies in newly diagnosed type I diabetes Saudi children, and their non-diabetic siblings. We studied 69 newly diagnosed type 1 diabetic Saudis (35 girls, 34 boys), 60 non-diabetic siblings (1 to 17 years), and 42 age- and sex-matched controls not having type 1 diabetes. Their sera were tested for insulin autoantibodies (IAA), antibodies to glutamic acid decarboxylase (GAD65A) and protein tyrosine phosphatase-2 antibodies (IA-2) using ¹²⁵I radioimmunoassays. Fifty-two percent of patients were significantly positive for IAA compared to controls (4.9 %). A total of 50.7 % of patients were also significantly positive for IA-2 compared to controls (4.8 %) (p < 0.0001). GAD65 antibody was detected in 81.2 % of the patients and in none of the controls (p < 0.0001). Both IAA and IA-2 are significantly higher among younger age group, unlike GAD65A, which is significantly higher among older age groups. In non-diabetic siblings, the frequencies of IAA, IA-2, and GAD65A were 6.8, 5.2, and 10.2 % (0.039), respectively, which were higher than in controls. IAA and IA-2 titers were significantly high among younger age group (<0.027), and GAD65A is significantly higher among older age group. A total of 21.6 % of diabetics were positive for all the three antibodies and 3.4 % in siblings. A total of 35.3 % were positive for two antibodies and none in siblings, while 39.2 % were positive for one antibody and 11.9 % in siblings. The combined GAD65 and IA-2 was positive in 81.3 % of young age, 80 % of middle, and in 100 % of the old age group. The screening of type 1 diabetes among Saudis showed the presence of diabetes-related antibodies in 96.1 % of all newly diagnosed patients, compared to 11.9 % of controls (non-diabetic siblings). IAA and IA-2 were significantly higher among younger age groups; GAD65A was significantly higher among older age. The combined GAD65 and IA-2 tests can be considered as a sensitive marker for predicting the occurrence of the disease in individuals at risk of type 1 diabetes in Saudis.