Esper Boel

A novel radioligand binding assay to determine diagnostic accuracy of isoform-specific glutamic acid decarboxylase antibodies in childhood iDDM

SummaryInsulin-dependent diabetes mellitus (IDDM) is associated with autoreactivity against GAD but the diagnostic sensitivity (positivity in disease) and specificity (negativity in health) of isoform-specific GAD antibodies have yet to be defined in assay systems suitable for screening large number of samples. One set of IDDM patient (n=10) and control (n=50) standard sera were used to develop quantitative antibody assays with in vitro synthesized recombinant 35S-methionine-labelled GAD65 and GAD67, respectively, and protein A-Sepharose to separate free from antibody-bound ligand. Binding levels were not normally distributed (p<0.0001) and therefore, the diagnostic accuracy of GAD antibodies was analysed by the ROC plots in population-based, consecutively-diagnosed, recent onset, 0–14 year-old patients (n=105), and matched, healthy control subjects (n=157). The ROC plots showed that the diagnostic sensitivity of GAD65 antibodies was 77% and the specificity 92% compared with 8% and 98%, respectively for GAD67 antibodies. In the IDDM sera, GAD65 and GAD67 antibodies were concordant in 7% (6 of 81) and GAD65 antibodies and ICA in 89% (72 of 81) without a correlation between the autoantibody levels. Autoantibodies to recombinant human islet GAD65 are specific and sensitive markers for childhood IDDM in this immunoassay with in vitro synthesized 35S-methioninelabelled recombinant GAD.

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.

Quantitative assay using recombinant human islet glutamic acid decarboxylase (GAD65) shows that 64K autoantibody positivity at onset predicts diabetes type.

At and before onset, most insulin-dependent diabetics (IDDM) have islet GAD65 autoantibodies (GAD65Ab). Since IDDM also occurs in older patients where non-insulin-dependent diabetes is common, we studied GAD65Ab at onset to classify diabetes type. Our quantitative immunoprecipitation assay uses recombinant human islet GAD65 stably expressed in hamster fibroblasts. Electrophoretic mobility was identical to native islet GAD65. Like native antigen, recombinant GAD65 migrated as two bands during electrophoresis, but converted to one under stronger reduction. Immunoprecipitation was linear with respect to antibody or antigen concentration. In 120 population-based diabetic patients of all ages grouped by treatment at onset and after 18 mo, GAD65Ab were present in 70% on insulin (n = 37), 10% on oral agent (n = 62, P < 0.0001), 69% changing from oral agent to insulin (n = 16, P < 0.001), and 1 of 33 controls. 65% with GAD65Ab, versus 8% without, changed from oral agent to insulin (P < 0.01). The GAD65Ab quantitative index was remarkably stable, and only 2 of 32 patients changed antibody status during follow-up. Concordance between GAD65Ab and islet cell antibodies was 93%. Quantitative correlation was approximate but significant. This highly sensitive, quantitative, high capacity assay for GAD65Ab reveals treatment requirements better than clinical criteria, perhaps guiding immunomodulatory therapy.

Two different types of intervening sequences in the glucoamylase gene from Aspergillus niger.

One single glucoamylase gene could be identified in the chromosomal DNA of Aspergillus niger by Southern blot analysis. This glucoamylase gene was isolated from a genomic library of A. niger DNA. The glucoamylase gene is situated on a 2.5‐kb EcoRI‐EcoRV fragment and contains five intervening sequences in the coding region. One 169‐bp intron is involved in differential mRNA processing leading to the two different glucoamylase enzymes G1 and G2; the other four introns are all very small ranging from 55 to 75 bp in length. One intron has a significant homology to the coding region which immediately follows, and it contains the internal conserved sequence TACTAAC, which is also found in yeast chromosomal gene introns, and is thought to participate in mRNA splicing. Two transcription initiation sites and a typical eukaryotic promoter region with TATAAT and CAAT boxes are located upstream from the gene.

Actions of phenylglycine analogs at subtypes of the metabotropic glutamate receptor family

The functional effects of phenylglycine analogs on metabotropic glutamate receptor (mGluR) subtypes mGluR1 alpha, mGluR2 and mGluR4 were examined. (S)-4-Carboxyphenylglycine (IC50 = 65 +/- 5 microM), (R,S)-alpha-methyl-4-carboxyphenylglycine (IC50 = 155 +/- 38 microM) and (S)-3-carboxy-4-hydroxyphenylglycine (IC50 = 290 +/- 47 microM) competitively antagonized glutamate-stimulated phosphoinositide hydrolysis in baby hamster kidney (BHK) cells stably expressing mGluR1 alpha. (S)-4-Carboxyphenylglycine and (R,S)-alpha-methyl-4-carboxyphenylglycine competitively antagonized glutamate-induced inhibition of forskolin-stimulated cAMP-formation in BHK cells stably expressing mGluR2 with IC50 values of 577 +/- 74 microM and 340 +/- 59 microM, respectively. (R,S)-4-carboxy-3-hydroxyphenylglycine, (R)-3-hydroxyphenylglycine and (S)-3-carboxy-4-hydroxyphenylglycine were agonists at mGluR2 with EC50 values of 48 +/- 5 microM, 451 +/- 93 and 97 +/- 12 microM, respectively. In parallel experiments, no activities of these phenylglycine analogs at mGluR4 were observed. The present report demonstrates that phenylglycine analogs possess differential functional activities at subtypes of the mGluR family.

Rhizomucor miehei triglyceride lipase is synthesized as a precursor.

ARhizomucor miehie cDNA library constructed inEscherichia coli was screened with synthetic oligonucleotides designed from knowledge of a partial amino acid sequence of the secreted triglyceride lipase (triacyl-glycerol acylhydrolase EC 3.1.1.3) from this fungus. Lipase-specific recombinants were isolated and their insert sequenced. Unlike characterized bacterial and mammalian triglyceride lipases, the fungal enzyme is synthesized as a precursor, including a 70 amino acid residue propeptide between the 24 amino acid residues of the signal peptide and the 269 residues of the mature enzyme. The precursor processing mechanism, which involves cleavage between a methionine and a serine residue, is unknown. By sequence comparison with other lipases, a serine residue involved in substrate binding was identified in the fungal lipase. The sequence around this residue is well-conserved among characterized lipases. Conservation of an intron in an isolated cDNA recombinant and immunoprecipitation of in vitro synthesizedR. miehei translation products indicates that the expression of the lipase gene might involve inefficient mRNA splicing.

Establishment of two continuous T-cell strains from a single plaque of a patient with mycosis fungoides.

SummaryFrom a plaque biopsy of a patient with mycosis fungoides, two different continuous cell lines were established by including both IL-2 and IL-4 in culture medium. Both continuous cell lines appeared with characteristic chromosome markers after approximately 40 cell population doublings. The initial karyotype recognized in T cells from the skin biopsy was 46,XY and the karyotypes of the continuous cell strains were 46,XY, -18, + i(18q) and another with multiple chromosome aberrations as described in Sezary T-cell leukemia. Phenotyping with monoclonal antibodies and T-cell receptor analysis indicates that the latter cell strain represents a minority of T-cells in the plaque. Due to its many chromosomal aberrations it probably represents the malignant cell, which may be a central cell in the immune stimulation taking place in the skin.

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.

Rhizomucor miehei triglyceride lipase is processed and secreted from transformedAspergillus oryzae

The cDNA encoding the precursor of theRhizomucor miehei triglyceride lipase was inserted in anAspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of theAspergillus oryzae α-amylase gene promoter and theAspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced intoAspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted fromA. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the nativeRhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted fromAspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

Identification of Glutamic Acid Decarboxylase Autoantibody Heterogeneity and Epitope Regions in Type I Diabetes

Glutamic acid decarboxylase (GAD) is an autoantigen of the islet cell antibodies (ICAs) present in type I diabetes. GAD autoantibodies are also found in patients with stiffman syndrome and in certain ICA-positive individuals who rarely develop diabetes on long-term follow-up. This latter subset of ICA has been termed restricted or beta-cell-specific ICA because the antibodies react with only the beta-cells of the islet. By immunoprecipitation of recombinant GAD65 and GAD67 protein and protein fragments, 83% of sera from individuals with new-onset diabetes or prediabetes (n = 30) had GAD65 autoantibodies, but only 26% had GAD67 autoantibodies. In contrast, all restricted ICA sera (n = 6) had both GAD65 and GAD67 autoantibodies. In both types of sera, the binding of GAD67 autoantibodies could be blocked by preincubation of the serum with GAD65 and GAD67, but the binding of GAD65 autoantibodies could not be blocked by preincubation with GAD67. The titer of GAD65 autoantibodies was much higher in the restricted ICA sera (titer > 1:1,000) than in the sera from individuals with new-onset diabetes or prediabetes (titer < 1:100) and was reflected by the greater amount of GAD65 protein immunoprecipitated by restricted ICA sera (2.61 ± 1.39 U) compared with sera from individuals with new-onset diabetes (0.51 ± 0.34 U). The restricted ICA sera immunoprecipitated equimolar amounts of GAD65 protein fragments, suggesting a non-conformational or linear epitope; epitope mapping localized the major epitope region to amino acids 361–442 and a second minor epitope region to amino acids 1–195. For the monoclonal antibody GAD6, the GAD65 epitope region localized to amino acids 529–585. In contrast, the sera from individuals with new-onset diabetes or prediabetes immunoprecipitated equal amounts of full-length GAD65 protein and a GAD65 fragment containing amino acids 188–585, but did not immunoprecipitate smaller GAD65 protein fragments, which suggests an epitope(s) dependent on protein conformation. These results suggest that subsets of GAD autoantibodies exist and indicate a heterogeneity in the immune response to GAD.