Glutamic Acid Decarboxylase Autoantibodies in Diabetes Mellitus

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

GLUTAMIC ACID DECARBOXYLASE AUTOANTIBODIES IN DIABETES MELLITUS Robert S. Schmidli, M.B., Ch.B. and Leonard C. Harrison M.D., D.Sc.

Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia

HISTORICAL NOTES

THE AUTOANTIGENS

Circulating autoantibodies to pancreatic islets of Langerhans in insulin-dependent diabetes mellitus (IDDM), termed islet cell antibodies (ICA), were first demonstrated in 1974 (Bottazzo et al., 1974). Although this stimulated considerable effort to identify target antigens, not until 1982 were sera from IDDM patients shown to immunoprecipitate a specific 64 kd islet protein from lysates of 35S-labeled rat islets (Baekkeskov et al., 1990). This 64 kd protein was further characterized (DeAizpurua et al., 1992b) and 64 kd antibodies were shown to be present prior to the onset of IDDM (Bottazzo, 1993). In 1988, glutamic acid decarboxylase (GAD) was shown to be an autoantigen in the rare neurological condition, stiffman syndrome (SMS) (Solimena and DeCamilli, 1991). The frequent association of SMS with organspecific autoimmune disease including IDDM (Blum and Jankovic, 1991) and the similarity of the molecular weights of GAD and the 64 kd antigen were the clues that led in 1990 to tlae identification of GAD as the 64 kd autoantigen in IDDM (Baekkeskov et al., 1990). Since its discovery in the central nervous system (CNS) in the 1950s, GAD has been studied extensively as the enzyme that catalyses the synthesis of the inhibitory neurotransmitter gamma amino butyric acid (GABA). GAD was first cloned from feline brain (Kaufman et al., 1986) and .was subsequently cloned from human brain and pancreas (Faulkner-Jones et al., 1995).

The two isoforms of GAD of predicted molecular weight 65 kd and 67 kd are known as GAD65 and GAD67. These are encoded by separate genes and share 65% identity and 80% similarity at the amino acid level; most of the differences are within the Nterminal 110 amino acids (Erlander et al., 1991) (Figure 1). Enzymatic activity requires binding of the cofactor pyridoxal 5'-phosphate (P-5-P) to a fourresidue motif situated near the C-terminus. GAD65 exists as an apoenzyme; whereas, GAD67 exists as a holoenzyme bound to P-5-P.

Tissue Expression The highest levels of GAD are in the brain (FaulknerJones et al., 1995) and significant GAD is expressed in the islets of Langerhans, pituitary, gonad, kidney, adrenal and liver in the rat. Because of its role as an autoantigen in IDDM, GAD has now been extensively studied in the pancreatic islets where different patterns of expression are found in the human, rat and mouse. At the protein level, human islets contain only GAD65; rat islets contain both forms and mouse islets predominantly GAD67, expressed at a lower level than in rat islets. In situ hybridization reveals that GAD65 and GAD67 gene expression is restricted to [3 cells in the rat and probably the mouse, but the lack of clear demarcation of [3 from o~ and other islet cells precludes such exact localization in human mouse (Faulkner-Jones et al., 1995). At the mRNA level, human islets contain GAD65 and GAD67 at a ratio of 200 to 1 (Cram et al., 1995); rat islets contain both

299

Figure 1. Amino acid (aa) sequence comparison, Coxsackie P2-C protein similarity and pyridoxal-5'-phosphate P-5-P binding site of GAD65 and GAD67.

forms, and GAD67 predominates in the mouse. GAD protein is detectable in several immortalized mouse ~cell lines, including the Simian virus 40 (SV40) transformed ~-cell lines 13-TC3 and [3HC. The [3TC3 cell line expresses both GAD mRNAs, GAD65 being more abundant. GAD67 mRNA is present in the SV40 transformed nonobese diabetic (NOD) mouse ~-cell line NIT-l, but not in the rat insulinoma line RINm5F nor the SV40 transformed hamster islet cell line HIT (Faulkner-Jones et al., 1995).

Recombinant GAD Recombinant GAD expressed in E. coli expression systems often lacks enzymatic activity, presumably due to improper folding. Production in eukaryotic hosts (such as Sf9 insect cells from baculovirus-based vectors) allows synthesis of large amounts of enzymatically active, immunoreactive GAD (Mauch et al., 1993). GAD can also be synthesized in small quantities by in vitro transcription and translation (Ujihara et al., 1994). Although IDDM sera react with native and recombinant GAD at similar frequencies (Schmidli et al., 1995), differences in antibody binding are documented. For example, the GAD65-specific monoclonal antibody GAD6 immunoprecipitates both GAD65 and GAD67 from brain (Butler et al., 1993), but only precipitates recombinant GAD65. The formation of heterodimers in tissue extracts is thought to give rise to coprecipitation of both GAD forms.

Methods of Purification Native GAD-purified by a combination of gel filtration, calcium phosphate gel and DEAE-Sephadex

300

chromatography was used to raise monoclonal antiGAD antibodies (Gottlieb et al., 1986), which are now used to purify GAD in a one-step immunoaffinity purification. Recombinant GAD, expressed in the baculovirus-Sf9 cell system, is purified by anion exchange chromatography or immunoaffinity chromatography (Moody et al., 1995). Recombinant GAD produced as a fusion protein with polyhistidines on the C-terminus is conveniently purified by nickelchelation affinity chromatography (Mauch et al., 1993). Purified, enzymatically active, baculovirusexpressed human GAD65 and GAD67 are available through the authors. E. coli or C. perfringens-derived GAD reported to be enzymatically active is available as a crude acetone powder or a partially purified powder from Sigma (St. Louis, Missouri, USA).

GAD Epitopes The patterns of antibody recognition of GAD in SMS and IDDM appear to differ. Generally, antibodies in sera from patients with SMS react with denatured GAD in immunoblots, whereas, those in IDDM sera only recognize GAD in its native form by immunoprecipitation (Baekkeskov et al., 1990). Additionally, GAD antibodies in SMS patients precipitate both GAD65 and GAD67; whereas, <20% of IDDM sera immunoprecipitate GAD67 (Butler et al., 1993). Furthermore, the titer of antibodies in SMS is generally higher than that in IDDM (Kim et al., 1994). Reported GAD65 antibody epitopes in SMS and IDDM are summarized in Figure 2. The study of GAD antibody epitopes is complicated by the presence of conformational epitopes and complex determinants spanning different regions of the molecule.

Figure 2. Summary of GAD65 epitopes in SMS and IDDM. Black rectangles denote conformational epitopes, grey rectangles denote linear epitopes.

AUTOANTIBODIES Pathogenetic Role Available evidence indicates that GAD antibodies do not have a pathogenic role in IDDM. In fact, GAD antibodies may signify relative protection from progressive ~-cell destruction. Animal Models. Transfer of peripheral blood mononuclear cells from patients with IDDM to mice with severe combined immunodeficiency disease (SCID) led to formation of GAD antibodies in some mice, but impairment of ~-cell function or evidence of islet cell damage did not occur (Petersen et al., 1993). Additionally, GAD antibodies are found in ICA-positive subjects without IDDM or impaired pancreatic ~-cell insulin release (Wagner et al., 1994). Further evidence of a "protective" effect of GAD antibodies is provided by a study in the NOD mouse. In this model of

spontaneous autoimmune IDDM, GAD antibodies occur at higher frequency and concentration and are detected at an earlier age in female mice with a lower incidence of diabetes (DeAizpurua et al., 1994). There are now a number of reports, however, which directly demonstrate a pathogenic role of GAD-reactive T cells in the NOD mouse. Intrathymic, intravenous or intraperitoneal injection of GAD65, and intraperitoneal injection of GAD67 delayed or prevented the appearance of insulitis and diabetes (Faulkner-Jones et al., 1995). This was accompanied by a reduction of Tcell proliferative responses to GAD in two of the studies (Tisch et al., 1993; Kaufman et al., 1993). Similar experiments were not undertaken in humans to determine whether GAD is a pathogenic autoantigen, because the presence of GAD in the central nervous system and the association of CNS disease with GAD autoimmunity raise serious concerns about tolerizing humans against GAD.

301

Human Disease. Despite the lack of direct evidence for a pathogenic role of GAD in human IDDM, partial evidence is provided by the existence of T-cell responses to GAD65 in newly diagnosed patients (Atkinson et al., 1992), to GAD67 in "at-risk" and newly diagnosed patients (Honeyman et al., 1993) and to a peptide sequence of the C-terminal of GAD65, aa473-555 (Lohman et al., 1994). Additionally, GADspecific cytotoxic T cells can be identified in IDDM patients (Panina-Bordignon et al., 1995). By contrast with SMS, all but one study shows that in IDDM, antibodies react only with conformational epitopes. IDDM sera immunoprecipitate fulllength GAD65 and a large fragment containing aa188--585, but do not precipitate smaller fragments (Ujihara et al., 1994). Using deletion mutants of GAD65 and five monoclonal antibodies derived from a patient with newly diagnosed IDDM, the presence of a midregion and C-terminal epitope was demonstrated in GAD65 (Richter et al., 1993). Antibody reactivity occurred against aa244--585, but further deletion of amino acids at the N-terminus did not affect antibody binding. The last 44 amino acids at the C-terminus were necessary for binding of three of the antibodies. Four of the five antibodies did not recognize the mutant missing aa363--422, which contains the P-5-P binding site. Binding by sera was abolished by deletion of aal--295, and deletion of 41 amino acids at the C-terminus prevents binding by four sera. Similar epitope patterns were found in a study in which chimeras of GAD65 and GAD67 were used to examine antibody binding (Daw and Powers, 1995); two distinct antibody specificities to GAD were found, one located in aa240-435 and the other in aa451--5 70. Mapping of overlapping fragments (-~100aa) encompassing human GAD67 by ELISA revealed a lower frequency of reactivity compared to GAD65 with 20% of newly diagnosed IDDM or "at risk" sera reacting with at least one GAD67 fragment. Most epitopes were in the mid- and C-terminal thirds of the protein, a region that shares a high degree of homology with GAD65. However, in another study (DeAizpurua et al., 1992b) using three fragments spanning mouse GAD67, reactivity by ELISA occurred with all fragments. The presence of a short region of similarity between GAD and the P2-C protein of coxsackievirus B 4 (Kaufman et al., 1992) (Figure 1) led to the suggestion that molecular mimicry might play a role in the genesis of GAD autoimmunity. Coxsackie virus B 4 can infect [3 cells and is epidemiologically linked

302

to IDDM in humans. The molecular mimicry theory is supported by the demonstration of T-cell proliferative responses to GAD65 peptides containing the shared sequence KXXPEVKEK, in "at-risk" and newly diagnosed IDDM subjects (Atkinson et al., 1994). Three out of three GAD peptide-reactive IDDM patients in this study also responded to the homologous coxsackie viral peptide; whereas, none of 13 controls did. In another study, immunization of mice with 16-mer coxsackie P2-C and GAD65 peptides containing the conserved sequence KXXPEVKEK generated T-cell responses only in NOD mice and not in nine other mouse strains (Tian et al., 1994). This was also the only strain in which cross-reactive T-cell recognition occurred. Thus, T-cell cross-reactivity in mice was restricted to the diabetes-associated NOD MHC class II allele, I-A g7. Direct proof of functional mimicry requires demonstration that pathogenic T-cell clones recognize the shared sequence. At the antibody level, molecular mimicry based on similarities between linear sequences is open to argument. Antibodies raised against either P2-C or GAD65 peptides cross-react with the other peptide in one report (Hou et al., 1994). Sera from coxsackie virus-infected mice contain antibodies to GAD65 protein and P2-C peptide, and some react to GAD65 peptide. Additionally, several IDDM sera react with GAD65 protein in addition to P2-C and GAD65 peptide. However, in another study with six humanderived, anti-GAD monoclonal antibodies which require the P2-C homology sequence of GAD65 for reactivity, no binding was found to a range of viral antigens derived from coxsackie B l--B6 (Richter et al., 1994). Furthermore, none of 15 coxsackie B 4positive human sera immunoprecipitated GAD65. Other infectious agents with sequence similarities to GAD include mycobacterial and human heat shock protein 60 (hsp60), Kunjin virus, Japanese encephalitis virus, Western Nile virus and Murray Valley encephalitis virus (Honeyman et al., 1993), but their significance is unknown.

Factors in Pathogenicity Subclasses. IgG subclasses may reflect the nature of T-cell responses and, therefore, the nature of immunemediated pathology. There are no published studies examining the IgG subclasses that react with GAD. Newly diagnosed IDDM patients have predominantly IgG1 _+ IgG3 antibodies to native brain GAD; on the other hand, ICA-positive relatives who do not pro-

gress to IDDM have predominantly IgG2 _+ IgG4 antibodies to GAD (Couper and Harrison, unpublished). Thus, the measurement of IgG subclass antibodies to GAD may be a practical way of refining the prediction of risk for progression to clinical IDDM. Genetics

Inheritance plays a major role in the susceptibility to IDDM, as indicated by twin studies in which pairwise concordance for IDDM ranges from 13--34% in monozygotic twins, compared to 2.5--5% in dizygotic twins. GAD antibodies may also be influenced by genetic factors. In a study of identical twins, 15% of siblings who were long-term discordant for IDDM were GAD Ab-positive (Bottazzo, 1993). Class II HLA genes are the major single genetic determinant of risk for IDDM, the highest risk haplotype in caucasoids being HLA DR3,4; DQ2,8, with "protection" being afforded by DR2 and DQ6 (Tait and Harrison, 1991). In one study, GAD antibodies were more prevalent (85%) in DR3, DR4 IDDM patients than in DR 3,4,X (48%) patients (Serjeantson et al., 1992). In a larger study, DQ2 was significantly more common in patients with GAD antibodies, and GAD antibodieswere detected in 64% of DQ2,8; 55% of DQ2,2 and 41% of patients with other HLA-DQB 1 alleles. The HLA-DQ2,8 association was more marked in those with onset of IDDM after the age of 14 years (Serjeantson et al., 1993). Methods of Detection

Among several methods to detect GAD antibodies, the most widely used are ELISA, radiobinding assay (RBA) and enzymatic immunoprecipitation (EIP). Indirect immunofluorescence (Genovese et al., 1992) and immunoprecipitation followed by immunoblotting (Baekkeskov et al., 1990) are used less frequently. In the ELISAs (DeAizpurua et al., 1992a; 1992b), purified recombinant or native GAD is bound to a microtiter plate, serum is incubated in the wells, and bound antibody is detected with an appropriate labeled secondary antibody. RBAs utilize radiolabeled recombinant (Hagopian et al., 1993a; Petersen et al., 1994; Grubin et al., 1994) or native (Rowley et al., 1992) GAD, which is precipitated with serum antibodies and detected by gamma or beta counting. 125I-labeled, immunoaffinity-purified pig brain GAD is often used as a source of native GAD for this type of assay.

Most assays that use recombinant GAD rely on biosynthetic labelling with 35S-methionine, but 125I can also be used successfully. There is little difference in assay sensitivity or specificity when recombinant and native GAD are used, or when 125I or 35S are used as the radiolabel (Schmidli et al., 1995). In the EIP assays (Baekkeskov et al., 1990; Martino et al., 1991; DeAizpurua et al., 1992b; Schmidli et al., 1994a), GAD is quantified after immunoprecipitation by enzymatic activity, usually measured as conversion of 14C-labeled glutamate to GABA and 14C02, the latter detected by adsorption to hyamine hydroxidesoaked filter paper disks. Assay performance was compared in two international GAD antibody workshops. Despite a wide range of methods, a surprisingly high degree of concordance was noted between laboratories. After standardization of assay sensitivity to give a specificity of 100%, the mean sensitivity for IDDM of RBAs was 66.7%, ELISAs 24.7% and EIPs 40.6%. Eight RBAs and one ELISA had a sensitivity >80% (Schmidli et al., 1995). RBA is, therefore, currently the preferred method, with 35S-labeled in vitro translated recombinant GAD being the most widely used source of antigen (Petersen et al., 1994; Grubin et al., 1994). Despite their obvious advantage of convenience, most currentlyavailable ELISAs are not sufficiently sensitive for applications such as preclinical screening for IDDM. There is no international standard measure of GAD antibodies at present, but dilutions of a standard serum and the monoclonal GAD65 antibody MICA6 generate a standard curve from which results can be interpolated, thus allowing standardization between laboratories.

CLINICAL UTILITY Disease Associations

GAD antibodies are specific for SMS and IDDM and are not detected in other disorders, except those, such as polyendocrine autoimmunity, which are associated with IDDM. GAD antibodies corroborate the clinical diagnosis of SMS, with a diagnostic sensitivity reported at 100% in well-characterized patients (Kim et al., 1994). As this condition coexists with IDDM, false-positive results may occur where IDDM is present and the diagnosis of SMS is in doubt. GAD antibodies may be useful for identifying adult-onset diabetic patients who have a slow onset of IDDM

303

typical of adults, variously referred to as latent autoimmune diabetes of adults, or "type 1.5" diabetes. Among newly diagnosed adult diabetic subjects, 65% of the GAD antibody-positive patients require a change from an oral agent to insulin within 18 months (Hagopian et al., 1993a). In another study, 76% of adult patients presenting with diabetes and impaired insulin response to glucagon had GAD antibodies, compared to 12% with a normal insulin response (Tuomi et al., 1993a). Antibodies to GAD are found in SMS and IDDM. The frequency of GAD antibodies in SMS was initially reported to be about 60% (Solimena and DeCamilli, 1991), but in a recent study (Kim et al., 1994), 100% of patients had GAD antibodies. The frequencies of GAD antibodies in IDDM range from 25% (Martino et al., 1991) to 79% (Hagopian et al., 1993a). Although this difference may reflect the genetic, racial (Serjeantson et al., 1992), age and gender (Schmidli et al., 1994b) composition of study groups, the lower figures are more likely due to less sensitive assays and the true positivity is probably 60--80%. In a study of 23 patients with polyendocrine autoimmunity, 21 who were ICA-positive were also positive for GAD antibodies (Wagner et al., 1994). Of these 21, six developed impaired first-phase insulin release in response to intravenous glucose, three developed IDDM, and the remaining 12 did not develop IDDM in 22--82 months follow-up. These results suggest that polyendocrine autoimmunity is associated with a high prevalence of GAD Ab, although these patients do not progress rapidly to IDDM. In our hands, however, only 2 out of 27 unselected patients with thyroid disease had elevated GAD antibody concentrations. No definite associations between GAD antibodies and diabetes complications are found. Despite an early report which suggested that diabetic neuropathy in a small number of patients was associated with high levels of GAD antibodies (Kaufman et al., 1992), subsequent studies have not shown this association (Tuomi et al., 1993b). ICA and GAD antibodies positivity are strongly correlated in preclinical and clinical IDDM subjects; in several studies, GAD antibodies were almost exclusively found in the presence of ICA (Martino et al., 1991; Schmidli et al., 1994b). Experimental evidence indicates that GAD represents a component of the ICA antigen. Incubation of ICA-positive sera with rat brain homogenate (Genovese et al., 1992), E. coli lysates containing recombinant human GAD65 (Atkinson et al., 1993) or purified recombinant human

304

islet GAD (Marshall et al., 1994) reduced or abolished ICA staining. In two of these studies, these "GADabsorbable" ICA associated with a low rate of progression to IDDM (Genovese et al., 1992; Atkinson et al., 1993). "GAD-adsorbable" ICA are associated with very high levels of GAD antibodies (Yu et al., 1994). Six anti-GAD monoclonal antibodies derived from an ICA-positive, newly diagnosed IDDM patient were designated monoclonal islet cell antibodies (MICA) 1--6 (Richter et al., 1993). The prevalence of GAD antibodies is also influenced by age, gender and perhaps race. In ICApositive first-degree relatives and newly diagnosed IDDM subjects, GAD antibodies are higher in postpubertal females than in males and prepubertal females (Schmidli et al., 1994b). The frequency of GAD antibodies in Asian IDDM patients is controversial; a lower prevalence of GAD antibodies was found in a small number of Hong Kong Cantonese, Korean and Japanese patients compared to Caucasians (Serjeantson et al., 1992), but in another study GAD antibodies were detected in over 80% of Japanese patients (Kawasaki et al., 1994). There is some evidence that high levels of GAD antibodies correlate with a lower risk of IDDM and a slower rate of decline in [~-cell function in ICApositive first-degree relatives (a group at-risk for IDDM). [3-cell destruction is considered to be mediated by cellular immunity, and in one study IDDM developed in ICA-positive relatives with evidence of cellular rather than humoral immunity (Harrison et al., 1993). In another study of ICA-positive relatives, GAD antibodies were inversely related to the loss of glucose-stimulated insulin release and the presence of insulin autoantibodies, a marker of high risk for IDDM (Yu et al., 1994). A number of potential preventive therapies for IDDM are being evaluated, including nicotinamide and induction of immune tolerance to insulin. Intervention therapy prior to diagnosis necessitates screening tests of high sensitivity and specificity. Currently, ICA is the most commonly used screening test for "preclinical" IDDM, but it is relatively labor-intensive, poorly reproducible and only semiquantitative. With the availability of recombinant GAD, large scale screening based on GAD antibodies is now possible. However, as the incidence of IDDM is relatively low in the general population, the presence of even a small number of false-positive cases lowers the positive predictive value of a screening test. This was the case when ICA were used to predict IDDM in the general

population. For this reason, screening studies continue to concentrate on first-degree relatives of patients with IDDM, who have an approximately 15-fold increase in risk for IDDM. No large-scale prospective study of the predictive value of GAD antibodies has yet been published in first-degree relatives or in an unselected population. In a retrospective study of pregnant women sampled during the antenatal period, GAD antibodies were detected in 82% of subjects who later developed IDDM and 36% who developed NIDDM, compared to 0% of 100 nonmatched controls. Several studies examined GAD antibodies in ICA or IAApositive first-degree relatives of patients with IDDM, a group in which 30-50% eventually develop IDDM. In two of these studies, the presence of GAD antibodies did not influence IDDM-free survival in firstdegree relatives with either ICA or IAA (Schmidli et al., 1994b) or ICA (Bingley et al., 1994). GAD antibodies in the absence of ICA are uncommon, reflecting the strong relationship between the two; only one GAD antibody-positive, ICA-negative subject was found in several separate studies of firstdegree relatives who were tested prior to onset of IDDM (Schmidli et al., 1994a; Thivolet et al., 1992).

GADAb

(nU/ml) 5000-

Plasmaphoresis

;'J

u u u 1 6 5 1 6 5 1u6u5 1 6 5 u1 16 65 5

2500-

Cyclophospharnide

b

lb

I

2b

i

30 16o

2bo

360

Days

Figure 3. Levels of GAD antibodies in a patient with SMS, treated with plasmapheresis followed by cyclophosphamide. 12 months. Although the effect of insulin therapy on GAD antibodies has not been formally examined, similar frequencies of GAD antibodies are found in recent-onset and established IDDM (Rowley et al., 1992; Schmidli et al., 1994a), suggesting that insulin therapy does not alter GAD antibody positivity.

CONCLUSION

Effect of Therapy There are few studies examining the effect of therapy on GAD antibodies. In one patient with SMS who was treated with plasmapheresis and cyclophosphamide (Figure 3), an immediate almost two-fold reduction in GAD antibody levels was followed by a further gradual decline to about a third of initial amount levels. However, GAD antibodies returned to pretreatment values when plasmapheresis was stopped, despite treatment with cyclophosphamide. This patient had muscular symptoms which did not alter when GAD antibodies were reduced by plasmapheresis (Schmidli and Harrison, unpublished). The effect of immunotherapy on GAD antibody levels in newly diagnosed IDDM has been examined in a placebocontrolled study of 132 patients; therapy with cyclosporin did not affect GAD antibodies over a period of

REFERENCES Atkinson MA, Kaufman DL, Campbell L, Gibbs KA, Shah SC, Bu DF, Erlander MG, Tobin AJ, Maclaren NK. Response of peripheral-blood mononuclear cells to glutamate decarboxylase in insulin-dependent diabetes. Lancet 1992;339: 458--459.

GAD antibodies are present in the majority of patients with SMS and IDDM. Although results from several studies indicate a pathogenic role for GAD in driving T-cell-mediated ~-cell destruction in the NOD mouse model, there is no direct evidence as yet that GAD is a pathogenic autoantigen in human IDDM. Nevertheless, GAD antibodies are a sensitive and specific marker of islet autoimmunity and may be used to identify humans "at-risk" for IDDM, including asymptomatic relatives of IDDM patients and a subgroup of people with adult-onset diabetes. The availability of technically simple GAD antibody assays could allow GAD antibodies to displace ICA as the "gold standard" immune marker of IDDM and become the initial screening test for preclinical IDDM. See also GAD IN STIFF-MAN SYNDROME, INSULIN AUTOANTIBODIES and ISLET CELL AUTOANTIBODIES.

Atkinson MA, Kaufman DL, Newman D, Tobin AJ, Maclaren NK. Islet cell cytoplasmic autoantibody reactivity to glutamate decarboxylase in insulin-dependent diabetes. J Clin Invest 1993;91:350--356. Atkinson MA, Bowman MA, Campbell L, Darrow BL, Kaufman DL, Maclaren NK. Cellular immunity to a determinant common to glutamate decarboxylase and coxsackie virus

305

in insulin-dependent diabetes. J Clin Invest 1994;94:21252129. Baekkeskov S, Aanstoot HJ, Christgau S, Reetz A, Solimena M, Cascalho M, Folli F, Richter-Olesen H, DeCamilli P. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature 1990;347:151-156. Bingley PJ, Christie MR, Bonifacio E, Bonfanti R, Shattock M, Fonte MT, Bottazzo GF, Gale EA. Combined analysis of autoantibodies improves prediction of IDD in islet cell antibody-positive relatives. Diabetes 1994;43:1304-- 1310. Blum P, Jankovic J. Stiff-person syndrome: an autoimmune disease. Mov Disord 1991;6:12--20. Bottazzo GF, Florin-Christensen A, Doniach D. Islet-cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiencies. Lancet 1974;2:1279--1283. Bottazzo GF. On the honey disease. A dialogue with Socrates. Diabetes 1993;42:778-800. Butler MH, Solimena M, Dirkx R Jr, Hayday A, De Camilli P. Identification of a dominant epitope of glutamic acid decarboxylase (GAD-65) recognized by autoantibodies in stiff-man syndrome. J Exp Med 1993;178:2097--2106. Cram DS, Faulkner-Jones B, Kun J, Harrison LC. Glutamic acid decarboxylase-67 (GAD67): expression relative to GAD65 in human islets and mapping of autoantibody epitopes. Endocrinology 1995;136:1111--1119. Daw K, Powers AC. Two distinct glutamic acid decarboxylase autoantibody specificities in IDD target different epitopes. Diabetes 1995;44:216--220. DeAizpurua HJ, Harrison LC, Cram DS. An ELISA for antibodies to recombinant glutamic acid decarboxylase in IDDM. Diabetes 1992a;41:1182-- 1187. DeAizpurua HJ, Wilson YM, Harrison LC. Glutamic acid decarboxylase autoantibodies in preclinical insulin-dependent diabetes. Proc Natl Acad Sci USA 1992b;89:9841--9845. DeAizpurua HJ, French MB, Chosich N, Harrison LC. Natural history of humoral immunity to glutamic acid decarboxylase in nonobese diabetic (NOD) mice. J Autoimmun 1994;7: 643--653. Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, Tobin AJ. Two genes encode distinct glutamate decarboxylases. Neuron 1991;7:91--100. Faulkner-Jones BE, French MB, Williamson W, Cram DS, Honeyman MC, Schmidli RS, Harrison LC. Expression of glutamic acid decarboxylase (GAD) and its role as an autoantigen in insulin-dependent diabetes. In: Shafrir E, editor. Lessons from Animal Diabetes. 1995:In press. Genovese S, Bonifacio E, McNally JM, Dean BM, Wagner R, Bosi E, Gale EA, Bottazzo GF. Distinct cytoplasmic islet cell antibodies with different risks for type 1 (insulin-dependent) diabetes mellitus. Diabetologia 1992;35:385-388. Gottlieb DI, Chang YC, Schwob JE. Monoclonal antibodies to glutamic acid decarboxylase. Proc Natl Acad Sci USA

1986;83:8808-8812. Grubin CE, Daniels T, Toivola B, Landin-Olsson M, Hagopian WA, Li L, Karlsen AE, Boel E, Michelsen B, Lernmark A. A novel radioligand binding assay to determine diagnostic accuracy of isoform-specific glutamic acid decarboxylase

306

antibodies in childhood IDDM. Diabetologia 1994;37:344-350. Hagopian WA, Karlsen AE, Gottsater A, Landin-Olsson M, Grubin CE, Sundkvist G, Petersen JS, Boel E, Dyrberg T, Lernmark A. Quantitative assay using recombinant human islet glutamic acid decarboxylase (GAD65) shows that 64K autoantibody positivity at onset predicts diabetes type. J Clin Invest 1993a;91:368--374. Hagopian WA, Michelsen B, Karlsen AE, Larsen F, Moody A, Grubin CE, Rowe R, Petersen J, McEvoy R, Lernmark A. Autoantibodies in IDDM primarily recognize the 65,000-M(r) rather than the 67,000-M(r) isoform of glutamic acid decarboxylase. Diabetes 1993b;42:631-636. Harrison LC, Honeyman MC, DeAizpurua HJ, Schmidli RS, Colman PG, Tait BD, Cram DS. Inverse relation between humoral and cellular immunity to glutamic acid decarboxylase in subjects at risk of insulin-dependent diabetes. Lancet 1993 ;341:1365-- 1369. Honeyman MC, Cram DS, Harrison LC. Glutamic acid decarboxylase 67-reactive T cells: a marker of insulin-dependent diabetes. J Exp Med 1993;177:535--540. Hou J, Said C, Franchi D, Dockstader P, Chatterjee NK. Antibodies to glutamic acid decarboxylase and P2-C peptides in sera from coxsackie virus B4-infected mice and IDDM patients. Diabetes 1994;43:1260-1266. Kaufman DL, McGinnis JF, Krieger NR, Tobin AJ. Brain glutamate decarboxylase cloned in lambda gt-ll: fusion protein produces gamma-aminobutyric acid. Science 1986; 232:1138-1140. Kaufman DL, Erlander MG, Clare-Salzler M, Atkinson MA, Maclaren NK, Tobin AJ. Autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes mellitus. J Clin Invest 1992;89:283-292. Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GS, Robinson P, Atkinson MA, Sercarz EE, Tobin AJ, Lehmann PV. Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 1993;366:69-72. Kawasaki E, Takino H, Yano M, Uotani S, Matsumoto K, Takao Y, Yamaguchi Y, Akazawa S, Nagataki S. Autoantibodies to glutamic acid decarboxylase in patients with IDDM and autoimmune thyroid disease. Diabetes 1994;43:80-86. Kim J, Namchuk M, Bugawan T, Fu Q, Jaffe M, Shi Y, Aanstoot HJ, Turck CW, Erlich H, Lennon V, Baekkeskov S. Higher autoantibody levels and recognition of a linear NH2-terminal epitope in the autoantigen GAD65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus. J Exp Med 1994;180:595--606. Li L, Hagopian WA, Brashear HR, Daniels T, Lernmark A. Identification of autoantibody epitopes of glutamic acid decarboxylase in stiff-man syndrome patients. J Immunol 1994;152:930-934. Lohmann T, Leslie RD, Hawa M, Geysen M, Rodda S, Londei M. Immunodominant epitopes of glutamic acid decarboxylase 65 and 67 in insulin-dependent diabetes mellitus. Lancet 1994 ;343:1607-1608. Marshall MO, Hoyer PE, Petersen JS, Hejnaes KR, Genovese S, Dyrberg T, Bottazzo GF. Contribution of glutamate

decarboxylase antibodies to the reactivity of islet cell cytoplasmic antibodies. J Autoimmun 1994;7:497-508. Martino GV, Tappaz ML, Braghi S, Dozio N, Canal N, Pozza G, Bottazzo GF, Grimaldi LM, Bosi E. Autoantibodies to glutamic acid decarboxylase (GAD) detected by an immunotrapping enzyme activity assay: relation to insulin-dependent diabetes mellitus and islet cell antibodies. J Autoimmun 1991;4:915--923. Mauch L, Seissler J, Haubruck H, Cook NJ, Abney CC, Berthold H, Wirbelauer C, Liedvogel B, Scherbaum WA, Northemann W. Baculovirus-mediated expression of human 65 kDa and 67 kDa glutamic acid decarboxylases in Sf9 insect cells and their relevance in diagnosis of insulindependent diabetes mellitus. J Biochem 1993;113:699--704. Moody AJ, Hejnaes KR, Marshall MO, Larsen FS, Boel E, Svendsen I, Mortensen E, Dyrberg T. Isolation by anionexchange of immunologically and enzymatically active human islet glutamic acid decarboxylase 65 overexpressed in Sf9 insect cells. Diabetologia 1995;38:14--23. Panina-Bordignon P, Lang R, van Endert PM, Benazzi E, Felix AM, Pastore RM, Spinas GA, Sinigaglia F. Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes. J Exp Med 1995; 181:1923-- 1927. Petersen JS, Marshall MO, Baekkeskov S, Hejnaes KR, HoierMadsen M, Dyrberg T. Transfer of type 1 (insulin-dependent) diabetes mellitus associated autoimmunity to mice with severe combined immunodeficiency (SCID). Diabetologia 1993;36:510--515. Petersen JS, Hejnaes KR, Moody A, Karlsen AE, Marshall MO, Hoier-Madsen M, Boel E, Michelsen BK, Dyrberg T. Detection of GAD65 antibodies in diabetes and other autoimmune diseases using a simple radioligand assay. Diabetes 1994;43:459--467. Richter W, Shi Y, Baekkeskov S. 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. Proc Natl Acad Sci USA 1993;90:2832-2836. Richter W, Mertens T, Schoel B, Muir P, Ritzkowsky A, Scherbaum WA, Boehm BO. Sequence homology of the diabetes-associated autoantigen glutamate decarboxylase with coxsackie B4-2C protein and heat shock protein 60 mediates no molecular mimicry of autoantibodies. J Exp Med 1994; 180:721--726. Rowley MJ, Mackay IR, Chen QY, Knowles WJ, Zimmet PZ. Antibodies to glutamic acid decarboxylase discriminate major types of diabetes mellitus. Diabetes 1992;41:548--551. Schmidli RS, Colman PG, Harrison LC. Do glutamic acid decarboxylase antibodies improve the prediction of IDD in first-degree relatives at risk for IDDM? J Autoimmun 1994a;7:873--879. Schmidli RS, DeAizpurua HJ, Harrison LC, Colman PG. Antibodies to glutamic acid decarboxylase in at-risk and clinical insulin-dependent diabetic subjects: relationship to age, sex and islet cell antibody status, and temporal profile. J Autoimmun 1994b;7:55-66.

Schmidli RS, Colman PG, Bonifacio E. Disease sensitivity and specificity of 52 assays for glutamic acid decarboxylase antibodies. The second international glutamic acid decarboxylase antibody workshop. Diabetes 1995;44:636--640. Serjeantson SW, Kohonen-Corish MR, Rowley MJ, Mackay IR, Knowles W, Zimmet P. Antibodies to glutamic acid decarboxylase are associated with HLA-DR genotypes in both Australians and Asians with type 1 (insulin-dependent) diabetes mellitus. Diabetologia 1992;35:996-1001. Serjeantson SW, Court J, Mackay IR, Matheson B, Rowley MJ, Tuomi T, Wilson JD, Zimmet P. HLA-DQ genotypes are associated with autoimmunity to glutamic acid decarboxylase in insulin-dependent diabetes mellitus patients. Hum Immunol 1993;38:97--104. Solimena M, DeCamilli P. Autoimmunity to glutamic acid decarboxylase (GAD) in stiff-man syndrome and insulindependent diabetes mellitus. TINS 1991;14:452-457. Tait BD, Harrison LC. Overview: the major histocompatibility complex and insulin dependent diabetes mellitus. Baillieres Clin Endocrinol Metab 1991;5:211--228. Thivolet CH, Tappaz M, Durand A, Petersen J, Stefanutti A, Chatelain P, Vialettes B, Scherbaum W, Orgiazzi J. Glutamic acid decarboxylase (GAD) autoantibodies are additional predictive markers of type 1 (insulin-dependent) diabetes mellitus in high risk individuals. Diabetologia 1992;35:570-576. Tian J, Lehman PV, Kaufman DL. T cell cross-reactivity between Coxsackievirus and glutamate decarboxylase is associated with a murine diabetes susceptibility allelle. J Exp Med 1994;180:1979--1984. Tisch R, Yang XD, Singer SM, Liblau RS, Fugger L, McDevitt HO. Immune response to glutamic acid decarboxylase correlates with insulitis in nonobese diabetic mice. Nature 1993;366:72--75. Tuomi T, Groop LC, Zimmet PZ, Rowley MJ, Knowles W, Mackay IR. Antibodies to glutamic acid decarboxylase reveal latent autoimmune diabetes mellitus in adults with a noninsulin-dependent onset of disease. Diabetes 1993a;42:359-362. Tuomi T, Zimmet PZ, Rowley MJ, Serjeantson SW, Mackay IR. Persisting antibodies to glutamic acid decarboxylase in type 1 (insulin-dependent) diabetes mellitus are not associated with neuropathy. Diabetologia 1993b;36:685. Ujihara N, Daw K, Gianani R, Boel E, Yu L, Powers AC. Identification of glutamic acid decarboxylase autoantibody heterogeneity and epitope regions in type I diabetes. Diabetes 1994;43:968--975. Wagner R, Genovese S, Bosi E, Becker F, Bingley PJ, Bonifacio E, Miles KA, Christie MR, Bottazzo GF, Gale EA. Slow metabolic deterioration towards diabetes in islet cell antibody positive patients with autoimmune polyendocrine disease. Diabetologia 1994;37:365-371. Yu L, Gianani R, Eisenbarth GS. Quantitation of glutamic acid decarboxylase autoantibody levels in prospectively evaluated relatives of patients with type I diabetes. Diabetes 1994:43: 1229--1233.

307