Isolation and characterization of human monoclonal autoantibodies to glutamic acid decarboxylase.

Production of human monoclonal autoantibodies to glutamic acid decarboxylase M(r) 65,000 (GAD65), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD65 autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD65 were produced using standard techniques. F(ab')2S from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to 125I-labelled GAD65 (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M(r) chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD65 of 2.2 x 10(9), 5.8 x 10(9), 1.3 x 10(10) mol/l(-1); affinities of the mouse monoclonals (n = 5) ranged from 1.1 x 10(8) to 5.4 x 10(10) mol/l(-1). The binding of each of the human monoclonals was inhibited by GAD6 F(ab')2 and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')2S suggesting that the epitopes for these antibodies were overlapping. Studies with GAD65/GAD67 chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to 125I-labelled GAD65. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD65 in the donor's serum and in the sera of patients with type-1 diabetes.

Increased expression of GAD65 and GABA in pancreatic beta-cells impairs first-phase insulin secretion.

The functional role of glutamate decarboxylase (GAD) and its product GABA in pancreatic islets has remained elusive. Mouse beta-cells express the larger isoform GAD67, whereas human islets express only the smaller isoform GAD65. We have generated two lines of transgenic mice expressing human GAD65 in pancreatic beta-cells (RIP7-hGAD65, Lines 1 and 2) to study the effect that GABA generated by this isoform has on islet cell function. The ascending order of hGAD65 expression and/or activity in beta-cells was Line 1 heterozygotes < Line 2 heterozygotes < Line 1 homozygotes. Line 1 heterozygotes have normal glucose tolerance, whereas Line 1 homozygotes and Line 2 heterozygotes exhibit impaired glucose tolerance and inhibition of insulin secretion in vivo in response to glucose. In addition, fasting levels of blood glucose are elevated and insulin is decreased in Line 1 homozygotes. Pancreas perfusion experiments suggest that GABA generated by GAD65 may function as a negative regulator of first-phase insulin secretion in response to glucose by affecting a step proximal to or at the K(ATP)(+) channel.

A non-cleavable mutant of Fas ligand does not prevent neutrophilic destruction of islet transplants.

BACKGROUND: Fas ligand (FasL) mediates apoptosis of susceptible Fas-expressing lymphocytes, and may contribute to the maintenance of peripheral tolerance. In transplantation models, however, artificial expression of FasL on cellular as well as islet transplants results in accelerated rejection by neutrophils. The mechanism of the neutrophilic response to FasL expression is unknown. FasL, like other members of the tumor necrosis factor family, is cleaved to a soluble form by metalloproteases. We tested the hypothesis that soluble FasL (sFasL) was responsible for neutrophil migration by creating a non-cleavable mutant of FasL. METHODS: Three mutants of FasL with serial deletions in the putative proteolytic cleavage site of human FasL were made using inverse polymerase chain reaction. The relative fractions of sFasL and membrane-bound FasL were assessed by Western blot and immunoprecipitation, as well as by cytotoxicity assay using Fas-expressing target cells. The fully non-cleavable mutant was transduced into murine islets as well as myoblasts and tumor cell lines, and tested in a murine transplantation model. RESULTS: Serial deletions in the putative metalloprotease site of FasL resulted in a fully non-cleavable mutant of FasL (ncFasL). Expression of ncFasL in tumor lines induced higher levels of apoptosis in Fas bearing targets than wild-type FasL. Transplantation of ncFasL-expressing islets under the kidney capsule of allogenic mice resulted in accelerated rejection identical to that seen with wild-type Fas ligand-expressing islets. Myoblasts and tumor cell lines expressing ncFasL also induced neutrophil infiltration. CONCLUSIONS: Membrane-bound Fas ligand is fully capable of inducing a neutrophilic response to transplants, suggesting an activation by Fas ligand of neutrophil chemotactic factors.

Peptide mapping and characterisation of glycation patterns of the glima 38 antigen recognised by autoantibodies in Type I diabetic patients.

AIMS/HYPOTHESIS: Glima 38 is an N-glycated neuroendocrine membrane protein of M(r) 38,000, which is recognised by autoantibodies in approximately 20% of patients with Type I (insulin-dependent) diabetes mellitus. The aim of this study was to characterise the carbohydrate moiety and generate peptide maps of glima 38. METHODS: Sera of high immunoreactivity to glima 38 were used to isolate 35-S methionine-labelled protein from betaTC-3 cells and a neuronal cell line GT1.7. Tunicamycin was used to inhibit N-glycation of glima 38 and define the core protein. The carbohydrate moiety was characterised for tunicamycin sensitivity, lectin binding and susceptibility to different endoglycosidases. The protein moiety was subjected to digestion by proteases to define peptide maps. RESULTS: The autoreactive epitopes in glima 38 recognised by Type I diabetic sera are conformational and independent of the carbohydrate moiety. Inhibition of N-glycation of glima 38 in vivo, shows a protein core of M(r) 22,000 in both pancreatic beta-(betaTC3) and neuronal (GT1.7) cell lines. The carbohydrate moieties in the two cell types are distinct but contain a similar amount of terminal sialic acid residues and at least five oligosaccharide chains Glima 38 binds Triticum vulgare and Ricinus communis I lectins. Endoproteinase treatment of the M(r) 22,000 core protein results in peptides of M(r) 4500 and M(r) 20,000 with Lys-C, and peptides of M(r) 4000 and M(r) 11,000-12,000 with Glu-C/V8 and Asp-N proteases. CONCLUSION/INTERPRETATION: The biochemical properties of glima 38 define it as a new autoantigen in Type I diabetes and provide a basis for its purification.

A dendritic cell line genetically modified to express CTLA4-IG as a means to prolong islet allograft survival.

BACKGROUND: Dendritic cells are potent antigen-presenting cells that bind allogeneic T cells. They are thus candidates for targeting immunoregulatory molecules to the alloreactive T cell compartment and suppressing the alloimmune response. METHOD: A dendritic cell line derived from the BALB/c mouse (H2d) was genetically modified to express the immunoregulatory molecule CTLA4-Ig. The ability of these dendritic cell transfectants to downregulate the alloimmune response was tested in an islet transplant model. Allogeneic C57Bl/6 (H2b) mice were rendered diabetic with streptozocin, and they received BALB/c islet (H2d) transplants. Mice were administered 25 million untransfected or CTLA4-Ig-transfected D2SC/1 cells i.v. on the day of islet transplantation and 6 days later[fnc]. RESULT: Mice treated with CTLA4-Ig-transfected D2SC/1 cells demonstrated prolonged allograft survival (mean = 20 days, median = 17 days, SD = 9.39) compared with mice treated with untransfected D2SC/1 cells (mean = 12 days, median = 11 days, SD=2.74) or untreated control mice (mean = 11 days, median = 11 days SD = 1.41). Third party allograft survival was not prolonged in mice receiving similar treatment. CONCLUSIONS: These results demonstrate that a genetically modified dendritic cell line can suppress the alloimmune response and prolong islet allograft survival in an allospecific manner. The findings also suggest that genetically modified dendritic cells may be useful in targeting alloreactive T cells and prolonging allograft survival.

The hydrophilic isoform of glutamate decarboxylase, GAD67, is targeted to membranes and nerve terminals independent of dimerization with the hydrophobic membrane-anchored isoform, GAD65.

GAD67, the larger isoform of the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase, is a hydrophilic soluble molecule, postulated to localize at nerve terminals and membrane compartments by heterodimerization with the smaller membrane-anchored isoform GAD65. We here show that the dimerization region in GAD65 is distinct from the NH(2)-terminal membrane-anchoring region and that a membrane anchoring GAD65 subunit can indeed target a soluble subunit to membrane compartments by dimerization. However, only a fraction of membrane-bound GAD67 is engaged in a heterodimer with GAD65 in rat brain. Furthermore, in GAD65-/- mouse brain, GAD67, which no longer partitions into the Triton X-114 detergent phase, still anchors to membranes at similar levels as in wild-type mice. Similarly, in primary cultures of neurons derived from GAD65-/- mice, GAD67 is targeted to nerve terminals, where it co-localizes with the synaptic vesicle marker SV2. Thus, axonal targeting and membrane anchoring is an intrinsic property of GAD67 and does not require GAD65. The results suggest that three distinct moieties of glutamate decarboxylase localize to membrane compartments, an amphiphilic GAD65 homodimer, an amphiphilic GAD65/67 heterodimer, tethered to membranes via the GAD65 subunit, and a hydrophilic GAD67 homodimer, which associates with membranes by a distinct mechanism.

The role of the synthetic enzyme GAD65 in the control of neuronal gamma-aminobutyric acid release.

We have studied GABAergic synaptic transmission in retinal ganglion cells and hippocampal pyramidal cells to determine, at a cellular level, what is the effect of the targeted disruption of the gene encoding the synthetic enzyme GAD65 on the synaptic release of gamma-aminobutyric acid (GABA). Neither the size nor the frequency of GABA-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were reduced in retina or hippocampus in GAD65-/- mice. However, the release of GABA during sustained synaptic activation was substantially reduced. In the retina both electrical- and K(+)-induced increases in IPSC frequency were depressed without a change in IPSC amplitude. In the hippocampus the transient increase in the probability of inhibitory transmitter release associated with posttetanic potentiation was absent in the GAD65-/- mice. These results indicate that during and immediately after sustained stimulation the increase in the probability of transmitter release is not maintained in GAD65-/- mice. Such a finding suggests a decrease in the size or refilling kinetics of the releasable pool of vesicles, and various mechanisms are discussed that could account for such a defect.

Glutamate decarboxylase and GABA in pancreatic islets: lessons from knock-out mice.

The GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) is expressed in pancreatic beta-cells and GABA has been suggested to play a role in islet cell development and function. Mouse beta-cells predominantly express the larger isoform of the enzyme, GAD67, and very low levels of the second isoform, GAD65. Yet GAD65 has been shown to be a target of very early autoimmune T-cell responses associated with beta-cell destruction in the non-obese diabetic (NOD) mouse model of Type 1 diabetes. Mice deficient in GAD67, GAD65 or both were used to assess whether GABA is important for islet cell development, and whether GAD65 is required for initiation of insulitis and progression to Type 1 diabetes in the mouse. Lack of either GAD65 or GAD67 did not effect the development of islet cells and the general morphology of islets. When GAD65-/-(129/Sv) mice were backcrossed into the NOD strain for four generations, GAD65-deficient mice developed insulitis similar to GAD65+/+ mice. Furthermore, at the low penetrance of diabetes in this backcross, GAD65-deficient mice developed disease at the same rate and incidence as wildtype mice. The results suggest that GABA generated by either GAD65 or GAD67 is not critically involved in islet formation and that GAD65 expression is not an absolute requirement for development of autoimmune diabetes in the NOD mouse.

High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase.

The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmunity affecting its major sites of expression, GABA-ergic neurons and pancreatic beta-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specific amino acid residues which form autoreactive B-cell epitopes in this molecule. Detailed mapping of 13 conformational epitopes, recognized by human monoclonal antibodies derived from patients, together with two and three-dimensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5'-phosphate-binding middle domain (residues 201-460) and to dialkylglycine decarboxylase in the C-terminal domain (residues 461-585). Six distinct conformational and one linear epitopes cluster on the hydrophilic face of three amphipathic alpha-helices in exons 14-16 in the C-terminal domain. Two of those epitopes also require amino acids in exon 4 in the N-terminal domain. Two distinct epitopes reside entirely in the N-terminal domain. In the middle domain, four distinct conformational epitopes cluster on a charged patch formed by amino acids from three alpha-helices away from the active site, and a fifth epitope resides at the back of the pyridoxal 5'-phosphate binding site and involves amino acid residues in exons 6 and 11-12. The epitopes localize to multiple hydrophilic patches, several of which also harbor DR*0401-restricted T-cell epitopes, and cover most of the surface of the protein. The results reveal a remarkable spectrum of human autoreactivity to GAD65, targeting almost the entire surface, and suggest that native folded GAD65 is the immunogen for autoreactive B-cells.

Increased anxiety and altered responses to anxiolytics in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase.

The larger isoform of the enzyme glutamate decarboxylase, GAD67, synthesizes >90% of basal levels of gamma-aminobutyric acid (GABA) in the brain. In contrast, the smaller isoform, GAD65, has been implicated in the fine-tuning of inhibitory neurotransmission. Mice deficient in GAD65 exhibit increased anxiety-like responses in both the open field and elevated zero maze assays. Additionally, GAD65-deficient mice have a diminished response to the anxiolytics diazepam and pentobarbital, both of which interact with GABA-A receptors in a GABA-dependent fashion to facilitate GABAergic neurotransmission. Loss of GAD65-generated GABA does not appear to result in compensatory postsynaptic GABA-A receptor changes based on radioligand receptor binding studies, which revealed no change in the postsynaptic GABA-A receptor density. Furthermore, mutant and wild-type animals do not differ in their behavioral response to muscimol, which acts independently of the presence of GABA. We propose that stress-induced GABA release is impaired in GAD65-deficient mice, resulting in increased anxiety-like responses and a diminished response to the acute effects of drugs that facilitate the actions of released GABA.

Differential protection in two transgenic lines of NOD/Lt mice hyperexpressing the autoantigen GAD65 in pancreatic beta-cells.

Although expressed at very low levels in islets of NOD mice, GAD65 is a candidate islet autoantigen. Two transgenic lines of NOD/Lt mice expressing high levels of human GAD65 from a rat insulin promoter were generated. Transgenes were integrated on proximal chromosome 15 of the A line and on the Y chromosome of the Y line. Transgenic A-line mice were obligate hemizygotes, since homozygous expression resulted in developmental lethality. A twofold higher level of hGAD65 transcripts in A-line islets from young donors was associated with higher GAD protein and enzyme activity levels. Y-line males developed diabetes at a similar rate and incidence as standard NOD/Lt males. In contrast, A-line mice of both sexes exhibited a markedly lowered incidence of diabetes. Insulitis, present in both transgenic lines, developed more slowly in A-line mice and correlated with a reduction in the ratio of gamma-interferon to interleukin-10 transcripts. Splenic leukocytes from young A-line donors transferred diabetes into NOD-scid recipients at a retarded rate compared with those from nontransgenic donors. Further, nontransgenic NOD T-cells transferred diabetes more slowly in NOD-scid recipients that were congenic for A-line transgenes as compared with standard NOD-scid recipients. Primed T-cell responses and spontaneous humoral reactivity to GAD65 failed to distinguish transgenic from nontransgenic mice. Quantitative differences in expression level or insertional mutagenesis are possible mechanisms of protection in the A line.

Local GABA circuit control of experience-dependent plasticity in developing visual cortex.

Sensory experience in early life shapes the mammalian brain. An impairment in the activity-dependent refinement of functional connections within developing visual cortex was identified here in a mouse model. Gene-targeted disruption of one isoform of glutamic acid decarboxylase prevented the competitive loss of responsiveness to an eye briefly deprived of vision, without affecting cooperative mechanisms of synapse modification in vitro. Selective, use-dependent enhancement of fast intracortical inhibitory transmission with benzodiazepines restored plasticity in vivo, rescuing the genetic defect. Specific networks of inhibitory interneurons intrinsic to visual cortex may detect perturbations in sensory input to drive experience-dependent plasticity during development.

Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction.

Fas ligand is believed to mediate immune privilege in a variety of tissues, including the eye, testis, and a subset of tumors. We tested whether expression of Fas ligand on pancreatic islets either following adenoviral or germline gene transfer could confer immune privilege after transplantation. Islets were infected with an adenoviral vector containing the murine Fas ligand cDNA (AdFasL), and were transplanted into allogenic diabetic hosts. Paradoxically, AdFasL-infected islets underwent accelerated neutrophilic rejection. The rejection was T cell and B cell independent and required Fas protein expression by host cells, but not on islets. Similarly, transgenic mice expressing Fas ligand in pancreatic beta cells developed massive neutrophilic infiltrates and diabetes at a young age. Thus, Fas ligand expression on pancreatic islets results in neutrophilic infiltration and islet destruction. These results have important implications for the development of Fas ligand-based immunotherapies.

Phosphorylation of serine residues 3, 6, 10, and 13 distinguishes membrane anchored from soluble glutamic acid decarboxylase 65 and is restricted to glutamic acid decarboxylase 65alpha.

GAD65, the smaller isoform of the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase is detected as an alpha/beta doublet of distinct mobility on SDS-polyacrylamide gel electrophoresis. Glutamic acid decarboxylase (GAD) 65 is reversibly anchored to the membrane of synaptic vesicles in neurons and synaptic-like microvesicles in pancreatic beta-cells. Here we demonstrate that GAD65alpha but not beta is phosphorylated in vivo and in vitro in several cell types. Phosphorylation is not the cause of the alpha/beta heterogeneity but represents a unique post-translational modification of GAD65alpha. Two-dimensional protein analyses identified five phosphorylated species of three different charges, which are likely to represent mono-, di-, and triphosphorylated GAD65alpha in different combinations of phosphorylated serines. Phosphorylation of GAD65alpha was located at serine residues 3, 6, 10, and 13, shown to be mediated by a membrane bound kinase, and distinguish the membrane anchored, and soluble forms of the enzyme. Phosphorylation status does not affect membrane anchoring of GAD65, nor its Km or Vmax for glutamate. The results are consistent with a model in which GAD65alpha and -beta constitute the two subunits of the native GAD65 dimer, only one of which, alpha, undergoes phosphorylation following membrane anchoring, perhaps to regulate specific aspects of GAD65 function in the synaptic vesicle membrane.

Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase.

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, is synthesized by two glutamate decarboxylase isoforms, GAD65 and GAD67. The separate role of the two isoforms is unknown, but differences in saturation with cofactor and subcellular localization suggest that GAD65 may provide reserve pools of GABA for regulation of inhibitory neurotransmission. We have disrupted the gene encoding GAD65 and backcrossed the mutation into the C57BL/6 strain of mice. In contrast to GAD67-/- animals, which are born with developmental abnormalities and die shortly after birth, GAD65-/- mice appear normal at birth. Basal GABA levels and holo-GAD activity are normal, but the pyridoxal 5' phosphate-inducible apo-enzyme reservoir is significantly decreased. GAD65-/- mice develop spontaneous seizures that result in increased mortality. Seizures can be precipitated by fear or mild stress. Seizure susceptibility is dramatically increased in GAD65-/- mice backcrossed into a second genetic background, the nonobese diabetic (NOD/LtJ) strain of mice enabling electroencephalogram analysis of the seizures. The generally higher basal brain GABA levels in this backcross are significantly decreased by the GAD65-/- mutation, suggesting that the relative contribution of GABA synthesized by GAD65 to total brain GABA levels is genetically determined. Seizure-associated c-fos-like immunoreactivity reveals the involvement of limbic regions of the brain. These data suggest that GABA synthesized by GAD65 is important in the dynamic regulation of neural network excitability, implicate at least one modifier locus in the NOD/LtJ strain, and present GAD65-/- animals as a model of epilepsy involving GABA-ergic pathways.

Immune reactivity of diabetes-associated human monoclonal autoantibodies defines multiple epitopes and detects two domain boundaries in glutamate decarboxylase.

Autoreactive islet cell Abs (ICA) accompany the pathogenic destruction of pancreatic beta cells in insulin-dependent diabetes mellitus (IDDM). Human monoclonal ICA (MICA 1-6), previously derived from a DR1/DR7-positive newly diagnosed diabetic patient, recognized the islet cell autoantigen glutamate decarboxylase 65 (GAD65) and defined two distinct conformational (MICA 1/3 and MICA 4/6) and one linear (MICA 2) autoimmune epitopes in this molecule. We have isolated 4 new ICA-reactive B cell lines, one from a DR4/DR11-positive newly diagnosed IDDM patient (MICA 7) and three from a DR3 homozygous patient with both IDDM and Graves' disease (MICA 8-10). Like MICA 1-6, MICA 7-10 are specific for GAD65, suggesting that GAD65-reactive B cells dominate the ICA response in IDDM. Comparative analysis of MICA 1-6 and MICA 7-10, using GAD65 mutants and blocking experiments, showed that MICA 7-10 define three novel conformational autoimmune epitopes in GAD65. Further structural analysis of the MICA 1-10 epitopes revealed two distinct and one overlapping region of epitope clusters. Thus, the C-terminal region, defined by amino acids 450 to 570, harbors the conformational MICA1/3 and MICA 7 epitopes as well as the linear epitope of MICA 2 (amino acids 506-531). The MICA 4/6 and MICA 10 epitopes are located in the middle region of the molecule defined by amino acids 245 to 449, whereas the N-terminal region contributes only to the MICA 8/9 epitopes (encompassed in amino acids 39-585). MICA 1-6, 7, and 8-10, derived from three IDDM patients of different HLA haplotypes, define six different epitopes in GAD65 and represent tools to determine the spectrum, possible HLA association, and temporal order of epitope recognition in IDDM.

Identification and characterization of glima 38, a glycosylated islet cell membrane antigen, which together with GAD65 and IA2 marks the early phases of autoimmune response in type 1 diabetes.

Immunoprecipitating IgG autoantibodies to glutamic acid decarboxylase, GAD65, and/or a tyrosine phosphatase, IA2, are present in the majority of individuals experiencing pancreatic beta cell destruction and development of type 1 diabetes. Here we identify a third islet cell autoantigen, a novel 38-kD protein, which is specifically immunoprecipitated with sera from a subset of prediabetic individuals and newly diagnosed type 1 diabetic patients. The 38-kD autoantigen, named glima 38, is an amphiphilic membrane glycoprotein, specifically expressed in islet and neuronal cell lines, and thus shares the neuroendocrine expression patterns of GAD65 and IA2. Removal of N-linked carbohydrates results in a protein of 22,000 Mr. Glima 38 autoantibodies were detected in 16/86 (19%) of newly diagnosed patients, including three very young children, who had a rapid onset of disease, and in 6/44 (14%) of prediabetic individuals up to several years before clinical onset. The cumulative incidence of GAD65 and glima 38 antibodies in these two groups was 83 and 80%, respectively, and the cumulative incidence of GAD65, glima 38, and IA2 antibodies in the same groups was 91 and 84%, respectively. GAD65, IA2, and glima 38 represent three distinct targets of immunoprecipitating IgG autoantibodies associated with beta cell destruction and type 1 diabetes.

Value of antibodies to GAD65 combined with islet cell cytoplasmic antibodies for predicting IDDM in a childhood population.

The value of a test for islet cell cytoplasmic antibodies together with a test for GAD65 antibodies to predict the subsequent development of diabetes over a period of 11.5 years was assessed in an open childhood population comprising 2,805 individuals. A single serum sample was obtained from each individual between 1975 and 1977 and screened for islet cell cytoplasmic antibodies for which eight individuals were positive (0.29%). During the average follow-up period of 11.5 years, four of eight islet cell antibody positive and three islet cell antibody negative individuals developed clinical diabetes. Sera from all individuals, who were islet cell antibody positive and/or developed diabetes (total of 11) and from 100 randomly selected control subjects were analysed for GAD65 antibodies. Six of eight islet cell antibody positive individuals were GAD65 antibody positive including all four who subsequently developed IDDM. Furthermore, one of the three islet cell antibody negative individuals who developed IDDM was GAD65 antibody positive both in 1976 and in 1989. Thus, a positive test for GAD65 antibodies alone correctly predicted diabetes in five of seven children, who developed the disease. Only one of the children, who developed diabetes was positive for insulin autoantibodies and this individual was also positive for islet cell cytoplasmic antibodies and GAD65 antibodies. One of the 100 control subjects was positive for GAD65 antibodies (1%). The results suggest that a single GAD65 antibody test may have a higher sensitivity for predicting IDDM than a test for islet cell cytoplasmic antibodies, but that a combined positive test for both antibodies increases the specificity for predicting IDDM over a period of 11.5 years.

Higher autoantibody levels and recognition of a linear NH2-terminal epitope in the autoantigen GAD65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus.

The smaller form of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in two human diseases that affect its principal sites of expression. Thus, destruction of pancreatic beta cells, which results in insulin-dependent diabetes mellitus (IDDM), and impairment of GABA-ergic synaptic transmission in Stiff-Man syndrome (SMS) are both characterized by circulating autoantibodies to GAD65. Anti-GAD65 autoantibodies in IDDM are predominantly directed to conformational epitopes. Here we report the characterization of humoral autoimmune responses to GAD65 in 35 SMS patients, of whom 13 (37%) also had IDDM. All SMS patients immunoprecipitated native GAD65 and the main titers were orders of magnitude higher than in IDDM patients. Furthermore, in contrast to the situation in IDDM, autoantibodies in 35 of 35 (100%) of SMS patients recognized denatured GAD65 on Western blots. Two major patterns of epitope specificity were identified on Western blots. The first pattern, detected in 25 of 35 SMS patients (71%), of whom 11 had IDDM (44%), was predominantly reactive with a linear NH2-terminal epitope residing in the first eight amino acids of GAD65. Nine of nine individuals who were HLA-haplotyped in this group carried an IDDM susceptibility haplotype and HLA-DR3, DQw2 was particularly abundant. The second pattern, detected in 10 of 35 patients (29%) of whom two had IDDM (20%), included reactivity with the NH2-terminal epitope plus strong reactivity with one or more additional epitope(s) residing COOH-terminal to amino acid 101. The second epitope pattern may represent epitope spreading in the GAD65 molecule, but may also include some cases of epitope recognition associated with IDDM resistant HLA-haplotypes. The principal NH2-terminal linear epitope in GAD65 distinguishes the reactivity of SMS and IDDM autoantibodies and may be a determinant of pathogenicity for GABA-ergic neurons. The greater magnitude and distinct specificity of the humoral response to GAD65 in SMS may reflect a biased involvement of the T helper cell type 2 (Th2) subset of CD4+ T cells and antibody responses, whereas IDDM is likely mediated by the Th1 subset of CD4+ T cells and cytotoxic T cell responses.