Kim Ry Hejnaes

Detection of GAD65 Antibodies in Diabetes and Other Autoimmune Diseases Using a Simple Radioligand Assay

Autoantibodies to glutamic acid decarboxylase (GAD) are frequent at or before the onset of insulin-dependent diabetes mellitus (IDDM). We have developed a simple, reproducible, and quantitative immunoprecipitation radioligand assay using as antigen in vitro transcribed and translated [35S]methionine-labeled human islet GAD65. By using this assay, 77% (77 of 100) of serum samples from recent-onset IDDM patients were positive for GAD65 antibodies compared with 4% (4 of 100) of serum samples from healthy control subjects. In competition analysis with unlabeled purified recombinant human islet GAD65, binding to tracer was inhibited in 74% (74 of 100) of the GAD65-positive IDDM serum samples compared with 2% of the control samples. The levels of GAD antibodies expressed as an index value relative to a standard serum, analyzed with or without competition, were almost identical (r = 0.991). The intra- and interassay variations of a positive control serum sample were 2.9 and 7.6%, respectively (n = 4). The frequency of GAD antibodies was significantly higher with IDDM onset before the age of 30 (80%, 59 of 74) than after the age of 30 (48%, 10 of 21) (P < 0.01). The prevalence of islet cell antibodies showed a similar pattern relative to age at onset. Because simultaneous occurrences of multiple autoimmune phenomena are common, we analyzed sera from patients with other autoimmune diseases. The frequency of GAD antibodies in sera positive for DNA autoantibodies (8% [2 of 25] and 4% [1 of 25] in competition analysis) or rheuma factor autoantibodies [12% (4 of 35) and 3% (1 of 35) in competition analysis] was not different from that in control samples. In contrast, in sera positive for ribonucleoprotein antibodies the frequency of GAD antibodies was significantly increased (73% [51 of 70] and 10% [7 of 70] in competition analysis [P < 0.025]). In conclusion, even large numbers of serum samples can now be tested for GAD65 antibodies in a relatively short time, allowing screening of individuals without a family history of IDDM for the presence of this marker.

Differential Expression of Glutamic Acid Decarboxylase in Rat and Human Islets

The GABA synthesizing enzyme GAD is a prominent islet cell autoantigen in type I diabetes. The two forms of GAD (GAD64 and GAD67) are encoded by different genes in both rats and humans. By in situ hybridization analysis of rat and human pancreases, expression of both genes was detected in rat islets, whereas only GAD64 mRNA was detected in human islets. Immunocytochemical analysis of rat and human pancreatic sections or isolated islets with antibodies to GAD64 and GAD67 in combination with antibodies to insulin, glucagon, or SRIF confirmed that a GAD64 and GAD67 expression were β-cell specific in rat islets. In contrast, only GAD64 was detected in human islets and was, in addition to β-cells, also surprisingly localized to some α-cells, δ-cells, and PP-cells. In long-term (4 wk) monolayer cultures of newborn rat islet cells, GAD64 expression remained β-cell specific as observed in vivo, whereas GAD67 was localized not only to the β-cells but also in the α-cells and δ-cells. A small but distinct fraction of GAD positive cells in these monolayer cultures did not accumulate GABA immunoreactivity, which may indicate cellular heterogeneity with respect to GABA catabolism or GAD enzyme activity. In a rat insulinoma cell line (NHI-6F) producing both glucagon and insulin depending on the culture conditions, GAD64 expression was detected only in cultures in which the insulin producing phenotype dominated. In conclusion, these data demonstrate that the two GAD isoforms are differentially expressed in rat and human islets but also that the expression differs according to culture conditions. These findings emphasize the need to consider both the species and culture conditions of islets.

New strategy for the design of ligands for the purification of pharmaceutical proteins by affinity chromatography

A new approach for the identification of ligands for the purification of pharmaceutical proteins by affinity chromatography is described. The technique involves four steps. Selection of an appropriate site on the target protein, design of a complementary ligand compatible with the three-dimensional structure of the site, construction of a limited solid-phase combinatorial library of near-neighbour ligands and solution synthesis of the hit ligand, immobilisation, optimisation and application of the adsorbent for the purification of the target protein. This strategy is exemplified by the purification of a recombinant human insulin precursor (MI3) from a crude fermentation broth of Saccharomyces cerevisiae.

Isolation by anion-exchange of immunologically and enzymatically active human islet glutamic acid decarboxylase 65 overexpressed in Sf9 insect cells

SummaryThe enzymel-glutamic acid decarboxylase is a major autoantigen of the beta cell. Autoantibodies against this enzyme are observed before the onset of insulin-dependent diabetes mellitus (IDDM) in man and may be of predictive value. There is evidence that this enzyme is involved in the development of autoimmune diabetes in animals. In order to facilitate the investigation of the role ofl-glutamine acid decarboxylase in IDDM, we expressed the 65 kDa isoform of human isletl-glutamic acid decarboxylase in insect cells using a baculovirus-based vector. The material was expressed at high levels (up to 50 mg/l of cells). Partially purified metabolically labelledl-glutamic acid decarboxylase bound to immunoglobulins in the sera from 20 of 49 subjects with newly-diagnosed IDDM. The enzyme was isolated in high yields (up to 26 mg/l cell culture) with fully maintained enzymatic activity by either ion-exchange chromatography or immunoaffinity chromatography. Purifiedl-glutamic acid decarboxylase inhibited the binding of radioactivel-glutamic acid decarboxylase, prepared by in vitro translation of mRNA, to immunoglobulins in the sera of subjects with IDDM. Recombinant human isletl-glutamic acid decarboxylase, isolated from Sf9 cells, is a suitable material for the large scale investigation of the utility of this enzyme in the prediction and prevention of autoimmune diabetes. [Diabetologia (1995) 38: 14–23

Cloning and expression of an interleukin-1β precursor and its conversion to interleukin-1β

A gene coding for a N‐terminal precursor of interleukin‐1β (IL‐1β) was cloned and expressed in E. coli. The isolated Met‐Glu‐Ala‐Glu‐IL‐1β precursor was enzymatically converted to IL‐1β by means of dipeptidylaminopeptidase (DAP I). This method ensured a correct N‐terminal residue and the often observed expression of Met‐IL‐1β was thus avoided. The pure and physically homogeneous product exhibited the characteristic properties of natural IL‐1β. The in vitro biological activity was measured in the lymphocyte‐activating factor assay and was compared to that of natural IL‐1β isolated from stimulated monocyte culture using exactly the same purification procedure. The specific biological activity of both products was 2 × 10−8 U/mg indicating that the recombinant product exhibits full biological activity.

Transfer of type 1 (insulin-dependent) diabetes mellitus associated autoimmunity to mice with severe combined immunodeficiency (SCID)

SummaryPancreatic beta-cell destruction and development of Type 1 (insulin-dependent) diabetes mellitus are associated with circulating islet cell antibodies. Mice with severe combined immunodeficiency (SCID mice) were reconstituted with peripheral blood mononuclear cells from Type 1 diabetic patients, one who was antibody positive and one antibody negative, and from healthy individuals. Reconstituted mice were subsequently immunized with rat islets in incomplete Freunds adjuvant or adjuvant alone. Seventeen mice received peripheral blood mononuclear cells obtained at three different time points from the islet cell antibody positive patient. Before immunization with rat islets two mice developed antibodies to glutamic acid decarboxylase, a major target for antibodies in Type 1 diabetes, whereas none were positive for cytoplasmic islet cell antibodies. Following immunization with rat islets, glutamic acid decarboxylase antibodies were detected by immunoprecipitation in three additional mice, two of which also became positive for cytoplasmic islet cell antibodies. Of 22 mice which received peripheral blood mononuclear cells from either the islet cell antibody negative patient (n = 5) or from two healthy individuals (n = 17), none were positive for islet cell autoantibodies before or after immunization. None of the islet cell antibody positive mice became hyperglycaemic, showed impaired glucose tolerance or islet cell damage when studied 40 days after immunization (i.e. 100 days after reconstitution). In conclusion these results show that human B lymphocytes producing diabetes-associated autoantibodies can be transferred to SCID mice and remain antigen sensitive, but also that autoantibodies alone are not sufficient to induce beta-cell destruction.

T cell proliferative responses to glutamic acid decarboxylase-65 in IDDM are negatively associated with HLA DR3/4

Based on studies in spontaneously non-obese diabetic (NOD) mice, it has been suggested that the Mr 65,000 isoform of glutamic acid decarboxylase (GAD65) is of major importance in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). In humans, antibodies to GAD65 are present before and at onset of the disease and in vitro T cell reactivity to GAD has also been reported. To further characterize the T cell recognition of GAD65, we incubated peripheral blood mononuclear cells from 45 newly diagnosed IDDM patients with purified recombinant human islet GAD65 and correlated the proliferative response with HLA DR haplotype and the presence of GAD65 autoantibodies. Fifty healthy individuals were studied as controls. Of the patients, 49% showed proliferative responses to GAD65 in contrast to only 4% of the controls. T cell proliferation to GAD65 was significantly more frequent in patients not being HLA DR3/4 heterozygous (19/29, 66%) as compared to HLA DR3/4 heterozygous patients (3/16, 19%) (p < 0.01). The difference was most pronounced in females with 64% (9/14) of the HLA non-DR3/4 patients being positive compared to none (0/6) of the HLA DR3/4 patients (p < 0.05). The overall frequency of GAD65 autoantibodies was 71% (32/45) with a similar distribution between patients with HLA DR3/4 (10/16, 63%) and HLA non-DR 3/4 (22/29, 76%). There was no correlation between levels of the T and B cell responses to GAD65 (r = 0.24). In conclusion, we find a proliferative T cell response to GAD65 in approximately 50% of recent onset IDDM patients and unexpectedly find the majority of responders to be HLA non-DR 3/4 heterozygous patients. No difference was observed in B cell responsiveness between the two HLA groups.

Characterization of the three 125I-iodination isomers of human insulin-like growth factor I (IGF1)

Human insulin-like growth factor I (IGF1) was labeled with 125I and the resulting mixture of iodination isomers was separated by reverse-phase HPLC. Three major radioactive peaks were isolated and identified by sequencing as the expected three monoiodinated species. The ranking of the affinities of the three isomers for the human IGF1 receptor was found to be Tyr24(125I) > Tyr31(125I) >> Tyr60(125I). The Tyr31(125I) isomer was shown to have an affinity similar to that of unlabeled IGF1 and is thus the tracer of choice for IGF1. The tracers were stable upon storage at -20 degrees C for at least 3 months.

Regulation of GAD expression in rat pancreatic islets and brain by γ-vinyl-GABA and glucose

Summary Glutamic acid decarboxylase (GAD) is an important autoantigen in insulin-dependent diabetes mellitus (IDDM), but little is known about its regulation and function in islet cells. We investigated the effects of the GABA-transaminase inhibitor γ-vinyl-GABA (GVG) on GAD expression in rat islets and brain in vitro and in vivo. In islets incubated in high glucose culture medium there was an increase in GAD activity, GAD65 and GAD67 protein levels compared to low-glucose conditions; however, even in high glucose, GVG still significantly suppressed GAD activity and GAD67 expression. Our observations suggest that glucose and GVG act on GAD in islets through different mechanisms. Quantitative immunohistochemistry of pancreatic sections from rats treated with GVG in vivo using novel monoclonal antibodies specific for GAD65 and GAD67, showed a decrease in GAD67 expression (p < 0.005) relative to untreated rats. The effects of GVG on rat pancreatic islets were very similar to those observed in brain of rats treated with GVG in vivo. In homogenates of cerebral tissue from GVG treated rats containing both membrane-bound and soluble protein GAD67 levels were significantly decreased while GAD65 levels were not significantly changed compared to untreated rats. In contrast, in homogenates of cerebral tissues containing only soluble cytosolic protein, GVG-treatment was also significantly found to decrease GAD65 levels. Taken together, these results suggest that GVG potentially could be of use to decrease GAD expression in islet cells and consequently to deviate/inhibit the autoimmune response against the beta cells seen in IDDM. [Diabetologia (1998) 41: 530–535]

Stabilization of Recombinantly Expressed Proteins

Downstream purification of recombinant proteins has become an increasingly challenging issue. Even very fragile and complex proteins are now expressed in heterologous systems, and the successive purification processes are subject to still higher demands of process control and documentation. Usually, downstream purification involves multiple, highly technological and rather complicated unit operations in which the proteins are subject to an environment differing greatly from what can be found in vivo. The protein structure is sensitive to such environmental changes, and protein stability becomes a crucial aspect of protein purification. Therefore, state-of-the-art purification strategies should focus not only on the highest homogeneity and purity, but also on the presence of unstable forms of the protein. Stability problems are sometimes amplified in the downstream purification process, because the proteins are produced in a nonnative environment. Three very common causes of the emergence of stability problems could be listed as follows: Recombinant proteins are usually expressed in hcterologous systems; obviously, the required folding and post-translational modifications must be performed by enzyme systems that d o not exactly represent those found in the original in vivo host system. Stabilizing environment found in vivo might not be present during downstream purification. Such stabilizing factors could be redox potentials, binding proteins, folding enzymes, cellular compartments, chaperons, stabilizing cofactors, membranes, and so forth. 3. During expression and purification, the protein concentration is usually much above the in vivo concentration. Thus, intermolecular interactions might influence the stability dramatically. This paper exemplifies how stability problems might be solved by an optimal design of the purification process. 1 .