Michael O. Marshall

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.

Sulphatide in islets of Langerhans and in organs affected in diabetic late complications: a study in human and animal tissue

SummarySulphatide has been found in rat islets of Langerhans and anti-sulphatide antibodies have been demonstrated in patients with insulin-dependent diabetes mellitus. Using a specific monoclonal antibody, Sulph I, directed against sulphatide, we investigated the in situ distribution of this glycolipid immunohistochemically; furthermore, the sulphatide concentration was determined in several organs and cells by thin-layer chromatography. The islets of Langerhans in all species examined, mouse, rat, pig, and monkey were intensively stained but exocrine tissue remained unlabelled. The sulphatide concentration in human islets was 150±46 pmol/100 islets. The only glycolipid-antigen detected was sulphatide. Regarding other tissues, sulphatide was found to be located in distal tubules in the kidney, peripheral nerves, distinct scattered spot-like structures in the choreoid layer of the eye, the ovum, and peripheral granulocytes. Sulph I injection in mice showed homing to kidney tubules. Lung, heart, liver, adrenal, spleen, lymph node and thymus were not stained by Sulph I. Thus, the distribution of sulphatide shows an association with organs known to be affected in diabetes, either initially or in late complications.

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

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.

The specificity of 1-acyl-sn-glycerol 3-phosphate acyltransferase in microsomal fractions from lactating cow mammary gland towards short, medium and long chain acyl-CoA esters.

The 1-acylglycerolphosphate actyltransferase from a microsomal fraction of lactating cow mammary gland was active towards acyl-CoAs of chain length C8-C18, but not towards butyryl-CoA or hexanoyl-CoA. The lack of activity towards butyryl-CoA and hexanoyl-CoA explains why butyric and hexanoic acid are largely excluded from the sn-2 position of triacylglycerols from cow milk. The chain length specificity of the acyltransferase was C16 greater than C14 greater than C12 greater than C10 greater than C8, which is essentially the same as the order with which the fatty acids are found at the sn-2 position of cow milk triacylglycerols. The specificity was not affected by the nature of the fatty acid (palmitic or oleic acid) at the sn-1 position of 1-acylglycerolphosphate, as predicted by the theory of noncorrelative acylation.

Factors influencing the in vitro activity of diacylglycerol acyltransferase from bovine mammary gland and liver towards butyryl-CoA and palmitoyl-CoA

1. Factors affecting the incorporation of butyrate relative to palmitate into the sn-3 position of triacylglycerol by the diacylglycerol acyltransferase from bovine mammary gland and liver was studied in vitro. 2. Butyrate incorporation from butyryl-CoA in the presence of palmitoyl-CoA was favoured by a high concentration of butyryl-CoA and by the presence of a long-chain acyl-CoA binding protein such as bovine serum albumin. 3. The relative activity of the enzyme from both tissues towards butyryl-CoA and palmitoyl-CoA was independent of the concentration of membrane-bound 1,2-dipalmitoylglycerol. 4. The significance of these results in relation to the unique presence of short-chain acids in ruminant milk triacylglycerols is discussed.

A quantitative immunocytochemical method for the measurement of islet cell cytoplasmic antibodies

A quantitative immunocytochemical method for the measurement of islet cell cytoplasmic antibodies has been developed. The method employs human or rat pancreas, a protein A-peroxidase/diaminobenzidine secondary antibody system and an independent measurement of islet total and exocrine mean integrated absorbance by scanning microdensitometry. Specific islet cell cytoplasmic antibody binding (islet total-exocrine mean integrated absorbance) was dependent on serum dilution and substrate reaction time. The detection limit was approximately 5 JDF units. Specific islet cell cytoplasmic antibody binding values with human and rat pancreas were similar. Specific islet cell cytoplasmic antibody binding (human pancreas) was greater (p less than 0.001) in sera from patients with newly diagnosed insulin dependent diabetes mellitus (0.119 +/- 0.086, n = 29) compared to normal sera (0.003 +/- 0.008, n = 29). Thus, the method has been validated and may be useful for measuring the blocking effect of potential antigens on specific islet cell cytoplasmic antibody.