Merrill J. Rowley

Antibodies to Glutamic Acid Decarboxylase Reveal Latent Autoimmune Diabetes Mellitus in Adults With a Non—Insulin-Dependent Onset of Disease

The classification of adults with diabetes mellitus can be invalidated by patients who initially present as NIDDM but who later become frankly insulin dependent. In some of these, the pathogenesis could be similar to that in IDDM, namely autoimmune destruction of the pancreatic β-cells. We studied 102 patients >35 yr of age at diabetes onset who had initially been nonketotic and non-insulin-dependent for ≥6 mo. They were classified according to glucagonstimulated C-peptide levels into an insulin-deficient group (n = 33) and a non-insulin-deficient group (n = 69). We measured antibodies to GAD, islet cell cytoplasm, thyroid antigens, and gastric parietal cells in both groups. Anti-GAD was significantly higher in the insulin deficient group, 76% (25 of 33), than in the non-insulin deficient group, 12% (8 of 69), and this difference was substantially greater than that shown for ICAs. Thus, in a proportion of adults who present with NIDDM, a slowly evolving autoimmune insulitis can be revealed by testing for anti-GAD. This could have important connotations not only for early intervention, but also for the correct classification of diabetes.

Latent Autoimmune Diabetes Mellitus in Adults (LADA): the Role of Antibodies to Glutamic Acid Decarboxylase in Diagnosis and Prediction of Insulin Dependency

Type 1 diabetes mellitus in adults may present in a manner similar to that of Type 2 diabetes but with a late development of insulin dependency. We studied 65 patients who presented with ‘adult‐onset’ diabetes after the age of 30 years. Of these patients, 19 required insulin therapy. The insulin‐treated patients were significantly younger, their onset of diabetes was at an earlier age, and their postprandial serum C‐peptide levels were lower than those of the non‐insulin‐treated group. Moreover, the insulin‐treated subjects had a higher mean concentration of antibodies to glutamic acid decarboxylase (GAD) (66.8 ± 10.2 units) than the patients who did not require insulin (9.9 ± 1.9 units) (p < 0.001) and their frequency of anti‐GAD positivity was 73.7% versus 4.3% (p < 0.001). Thus, among patients attending a diabetes clinic, the majority (73.7%) of subjects who presented with diabetes after 30 years of age and who subsequently required therapy with insulin, actually have the islet cell lesion of Type 1 diabetes which progresses at a slower tempo than in children. We conclude that testing for anti‐GAD in adult‐onset non‐obese diabetic patients should be a routine procedure in order to detect latent insulin‐dependency at the earliest possible stage, since this assay can assist in the correct classification of diabetes, and more appropriate therapy.

Antibodies to Glutamic Acid Decarboxylase Discriminate Major Types of Diabetes Mellitus

Insulin-dependent diabetes mellitus (IDDM) is marked by circulating antibodies to a 64,000-Mr islet cell antigen identified as glutamic acid decarboxylase (GAD). We describe a radioimmunoprecipitation assay with GAD isolated from pig brain. The sera tested were from 80 patients with IDDM including 26 with disease of recent onset and 54 with disease of longer duration (3–42 yr), 20 with non-insulin-dependent diabetes mellitus (NIDDM), and 55 nondiabetic subjects. Conventional assays for islet cell cytoplasmic antibodies were performed concurrently. The level of antibody in serum was expressed in units based on percentage reactivity of a standard reference serum. The frequency of antibody to GAD in IDDM was 69% in short-duration cases and 59% in long-duration cases. The latter was substantially higher than the frequency of islet cell cytoplasmic antibody. Antibodies to GAD were elevated (means ± 3 SD) in 5% NIDDM cases and in none of the nondiabetic subjects. A simple laboratory test with a defined autoantigen has substantial implications for population screening and early diagnosis of IDDM and for better understanding of its pathogenesis.

GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop.

Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5′-phosphate–dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.

Anti-glutamate decarboxylase and other antibodies at the onset of childhood IDDM: a population-based study.

SummarySera obtained at diagnosis from 273 children (0–14 years) with insulin-dependent diabetes mellitus (IDDM) were studied to compare different autoantibody levels. The subjects comprise 75% of all incident cases in New South Wales, Australia, for a 2-year period (ascertainment >99% complete). Antibodies against glutamate decarboxylase were measured by radioimmunoprecipitation, insulin autoantibodies (on 176 sera collected within 4 days of initiation of insulin therapy) by radioimmunoassay, thyroid peroxidase and antigliadin IgA antibodies by enzyme-linked immunoassay, and anti-endomysial IgA and islet cell antibodies by indirect immunofluorescence. Reference ranges for anti-glutamate decarboxylase and insulin autoantibodies were determined in a group of non-diabetic children. Of the sera 69% were positive for anti-glutamate decarboxylase, 65% for insulin autoantibodies, 71% for islet cell antibodies (⩾20 Juvenile Diabetes Foundation units), 10% for anti-thyroid peroxidase, 2.6% for antigliadin and 3.0% for anti-endomysial antibodies. Islet cell antibodies and insulin autoantibodies were both negative in 13.7% of the sera, while only 5.8% were negative for all three of islet cell antibodies, insulin autoantibodies and anti-glutamate decarboxylase. There was a higher frequency of anti-glutamate decarboxylase among girls than boys (75% vs 63%, p=0.03) and a negative correlation between the level of insulin autoantibodies and age at diagnosis (r=−0.41, p<0.0001). A higher frequency of antithyroid peroxidase was found with increasing age (p=0.05). Higher titres of islet cell antibodies were associated with a higher frequency of both anti-glutamate decarboxylase (p<0.0001) and insulin autoantibodies (p=0.003). Five children (1.8%) with clear elevations of antigliadin and anti-endomysial antibodies were found to have asymptomatic coeliac disease by small bowel biopsy.

Pancreatic Islet Cell Cytoplasmic Antibody in Diabetes is Represented by Antibodies to Islet Cell Antigen 512 and Glutamic Acid Decarboxylase

The presence of serum islet cell cytoplasmic antibodies (ICAs) is a standard autoimmune marker for insulin-dependent diabetes mellitus (IDDM). The antigenic molecule(s) responsible for ICA has not been identified, although antibodies to the 65-kDa isoform of glutamic acid decarboxylase (GAD65) do contribute. We tested 129 IDDM sera for antibodies to ICA512 (anti-ICA512), antibodies to GAD (anti-GAD), and ICAs; we tested for inhibition of ICAs with purified recombinant ICA512 and sheep brain GAD; and we tested for immunofluorescence reactivity on COS7 cells transfected with cDNA clones encoding ICA512 and GAD65. The results were that anti-ICA512 antibodies contribute to ICA reactivity and that these, in combination with anti-GAD antibodies, account for most ICA reactivity in IDDM. Anti-ICA512 antibodies were present at a frequency of 51% in 61 patients with early-onset IDDM (age of onset ≤20 years) of short duration (≤1 month) but only in 9% of 68 patients with an onset age of >20 years and/or a disease duration of >1 month. The frequency of anti-GAD antibodies in these sera was similar irrespective of duration or age of onset. Anti-ICA512 and anti-GAD antibodies were demonstrable by indirect immunofluorescence on transfected COS7 cells, and ICA could be inhibited using either recombinant ICA512 or purified brain GAD. We conclude that anti-ICA512 and anti-GAD antibodies contribute to ICA reactivity and that anti-ICA512 antibodies account for the increased frequency of ICA reactivity in early-onset IDDM of short duration.

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.

The role of collagen antibodies in mediating arthritis

This review examines evidence that rheumatoid arthritis (RA) depends on autoimmunity to articular collagen, and mechanisms whereby autoantibodies to type II collagen contribute to disease development. Three major autoantigenic reactants have been identified in RA; the corresponding autoantibodies are rheumatoid factor (RF), antibodies to citrullinated peptide antigens (ACPA), citrullinated peptides (anti-CCP), and anti-type II collagen (anti-CII). Both RF and ACPA are well-validated and predictive markers of severe erosive RA, but cannot be linked to pathogenesis. By contrast, in various animal species immunized with CII there occurs an erosive inflammatory arthritis resembling that seen in human RA, together with antibodies to CII with an epitope specificity similar to that in RA. We discuss the well-known role of immune complexes in the induction of inflammation within the joint, and present recent data showing, additionally, that antibodies to CII cause direct damage to cartilage in vitro. The close resemblances between human RA and collagen-induced arthritis in animals suggest that autoimmunity, and particularly autoantibodies to CII, are important for both the initiation and perpetuation of RA in a dual manner: as contributors to the inflammation associated with immune complex deposition, and as agents with direct degradative effects on cartilage integrity and its repair.

Anti-neutrophil cytoplasm antibodies in rheumatoid arthritis.

Anti‐neutrophil cytoplasm antibodies (ANCA) occur occasionally in rheumatoid arthritis (RA), but their incidence and clinical significance have been unclear. In this study we have investigated 58 patients with RA. In 22 patients the disease was inactive and the remaining 36 with active disease were further subdivided into those without clinical evidence of vasculitis (26), those with cutaneous vasculitis (8) and those with systemic vasculitis (2). ANCA were demonstrated by indirect immunofluorescence in 10 of the 58 patients (17%), While both pcrinuclear (pANCA) and cytoplasmic (cANCA) staining were detected, pANCA were more common (70%). Neutrophil‐specific anti‐nuclear antibodies (ANNA) were demonstrated in a further eight sera (14%) and ANA were detected on Hep‐2 cells in 30 of the 58 sera (52%). ELISAs for the detection of anti‐myeloperoxidase and anti‐elastase antibodies were then established. Five sera with pANCA and five that contained ANNA were negative for both anti‐myeloperoxidase and anti‐elastase antibodies, suggesting other as yet unidentified cytoplasmic antigens as the target molecules. However, anti‐myeloperoxidase or anti‐elastase antibodies were found in four sera that had homogeneous or speckled ANA on both Hep‐2 cells and neutrophils. One serum contained both antibodies. The presence of ANCA detected by indirect immunofluorescence or of anti‐myeloperoxidase or anti‐elastase antibodies in these patients with RA was not associated with disease activity nor with the demonstration of cutaneous vasculitis or renal disease (P NS). A possible association with systemic vasculitis remains to be confirmed. There is an incomplete correlation between indirect immunofluorescence patterns and antibody specificity in ELISA systems.

The ethnic distribution of antibodies to glutamic acid decarboxylase: Presence and levels in insulin-dependent diabetes mellitus in Europid and Asian subjects

Our objective was to ascertain the frequency of antibodies to glutamic acid decarboxylase (GAD) in Europids and four Asian ethnic groups with insulin-dependent diabetes mellitus (IDDM) to gain insight into why the prevalence and incidence of IDDM varies so widely among ethnic and/or geographically diverse population groups. The subjects in this study were Europid (n = 49), Japanese (n = 16), Thai (n = 7), Korean (n = 21), and Chinese (n = 13) persons with IDDM with a duration ranging from 5 to 14 years. There were similar numbers of healthy controls matched for each ethnic group. A validated radioimmunoprecipitation assay used GAD from pig brain radiolabeled with 125I using chloramine T. Islet cell cytoplasmic antibodies measured by indirect immunofluorescence were expressed as Juvenile Diabetes Foundation units. The prevalence of antibodies to GAD, compared with Europids (63%), was much lower in all Asian populations with IDDM: Japanese (31%), Thai (29%), Korean (5%), and Chinese (27%). The mean level of antibodies to GAD, however, among diabetics from each population who gave a positive reaction, was similar. For all groups, the prevalence of antibodies to GAD was much higher than that of islet cell cytoplasmic antibodies. Almost all IDDM subjects positive for islet cell antibodies had antibodies to GAD, but the converse did not hold. A radioimmunoprecipitation assay for antibodies to GAD applied to serum from subjects with IDDM in various ethnic groups showed that Europids with IDDM had a much higher prevalence of such antibodies than did Asians. This held for all ethnic groups, and particularly Koreans. Thus, among different populations, there may be etiologic heterogeneity of IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)