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M J Rowley

Publications and source records attributed to M J Rowley.

At least 37 records · Page 2Linked to original sources
PubMed2000

Automated enzymatic mitochondrial antibody assay for the diagnosis of primary biliary cirrhosis.

Primary biliary cirrhosis is a progressive autoimmune disease that affects middle aged women, resulting in liver cirrhosis. We describe here an automated enzymatic mitochondrial antibody assay adapted for performance on laboratory analysers for the serological diagnosis of primary biliary cirrhosis. This assay detects the characteristic autoantibody directed against the 74kDa E2 subunit of the pyruvate dehydrogenase complex. Analysis of receiver operator characteristic curve data indicated that the automated enzymatic mitochondrial assay procedure discriminated clinically identified patients with primary biliary cirrhosis from normal subjects with a sensitivity of 83% and a specificity of 100%. This method compared favourably against a commercial ELISA method which had a sensitivity of 73% and a specificity of 100%. The automated enzymatic mitochondrial antibody assay is a high throughput assay of use for the routine diagnosis of patients with primary biliary cirrhosis with autoantibodies to the E2 subunit of the pyruvate dehydrogenase complex. The method is of potential value for economical and rapid screening to detect asymptomatic primary biliary cirrhosis in the at-risk segment of the population, namely middle aged women.

Autoantibodies↗
PubMed2000

Sex-determining region Y-related protein SOX13 is a diabetes autoantigen expressed in pancreatic islets.

The SOX (sex-determining region [SRY]-type high mobility group [HMG] box) family of transcription factors play key roles in determining cell fate during organ development. In this study, we have identified a new human SOX gene, SOX13, as encoding the type 1 diabetes autoantigen, islet cell antigen 12 (ICA12). Sequence analysis showed that SOX13 belongs to the class D subgroup of SOX transcription factors, which contain a leucine zipper motif and a region rich in glutamine. SOX13 autoantibodies occurred at a significantly higher frequency among 188 people with type 1 diabetes (18%) than among 88 with type 2 diabetes (6%) or 175 healthy control subjects (4%). Deletion mapping of the antibody epitopes showed that the autoantibodies were primarily directed against an epitope requiring the majority of the protein. SOX13 RNA was detected in most human tissues, with the highest levels in the pancreas, placenta, and kidney. Immunohistochemistry on sections of human pancreas identified SOX13 in the islets of Langerhans, where staining was mostly cytoplasmic. In mouse pancreas, Sox13 was present in the nucleus and cytoplasm of beta-cells as well as other islet cell types. Recombinant SOX13 protein bound to the SOX consensus DNA motif AACAAT, and binding was inhibited by homodimer formation. These observations-along with the known molecular interactions of the closely related protein, rainbow trout Sox23-suggest that SOX13 may be activated for nuclear import and DNA binding through heterodimer formation. In conclusion, we have identified ICA12 as the putative transcription factor SOX13 and demonstrated an increased frequency of autoantibody reactivity in sera from type 1 diabetic subjects compared with type 2 diabetic and healthy control subjects.

Amino Acid Sequence↗
PubMed1999

Antibodies to type II collagen and HLA disease susceptibility markers in rheumatoid arthritis.

OBJECTIVE: To seek associations between antibodies to native and denatured type II collagen (NCII and DCII) and HLA in rheumatoid arthritis (RA). METHODS: One hundred fourteen patients with clinically well-defined RA were HLA-DR and DQ typed. Those who were DR4 positive were subtyped for DRB1*0401-*0408 alleles by polymerase chain reaction using allele-specific oligonucleotide probes. Antibodies to human NCII and DCII (heat-denatured) were measured by enzyme-linked immunosorbent assay. The frequency of HLA alleles was compared in patients grouped according to the presence and absence of antibodies to NCII and DCII. RESULTS: Twenty-seven patients (24%) were positive for antibodies to NCII. There was a significant increase in the frequency of HLA-DR7 in anti-NCII-positive patients compared with anti-NCII-negative patients (30% versus 9%; P = 0.019) and a significant decrease in HLA-DR3 (7% versus 28%; P = 0.044). Repeating the analyses after excluding the 16 patients who were DR7 positive revealed a significant increase in the frequency of HLA-DR1 in anti-NCII-positive patients compared with anti-NCII-negative patients (63% versus 27%; P = 0.045). Moreover, antibodies to NCII were associated with the third hypervariability region susceptibility sequence QRRAA that is present in DRB1*0101, *0404, *0405, and *0408 (84% versus 47%; P = 0.0085); 24 of 27 anti-NCII-positive patients were positive for either DR7, DR1, or DRB1*0404 or *0408. Thirty patients (26%) were positive for antibodies to DCII. There was a significant increase in the frequency of HLA-DR3 in anti-DCII-positive patients compared with anti-DCII-negative patients (40% versus 18%; P = 0.028). CONCLUSION: The genetic associations between HLA-DR alleles and antibodies to CII in RA patients is in keeping with the collagen-induced arthritis model and implicates autoimmunity to CII as a major component in the multifactorial pathogenesis of RA.

Alleles↗
PubMed1999

Anti-idiotypic antibodies to anti-PDC-E2 in primary biliary cirrhosis and normal subjects.

Anti-idiotypic antibodies may regulate the immune system and influence pathogenic autoimmunity. We investigated idiotype-anti-idiotype interactions in sera of patients with primary biliary cirrhosis (PBC), normal subjects and animals immunized with pyruvate dehydrogenase complex (PDC) or its derivatives. IgG autoantibody to the E2 subunit of PDC (PDC-E2) was derived by affinity-purification from sera of 12 patients with PBC, and F(ab)2 was prepared (anti-PDC-E2-F[ab]2). This was used as a reactant by enzyme-linked immunosorbent assay (ELISA) with sera from patients with PBC, normal subjects, or immunized animals. Results were that IgG antibody to anti-PDC-E2-F(ab)2 was detectable at low concentration in 12 PBC sera (mean optical density [OD] +/- SD: 1.02 +/- 0.26), and also in 19 normal sera (mean OD +/- SD: 0.97 +/- 0. 35) using a serum dilution of 1:20; background OD was 0.09 to 0.10, whereas antisera from animals immunized with PDC or PDC-E2 were nonreactive. There was a significant inverse correlation (r = -.59, P =.04) between the levels of anti-PDC-E2 in PBC sera (but not normal sera), and anti-idiotypic antibody reactive with anti-PDC-E2-F(ab)2. Anti-idiotypic antibody existed as a complex with anti-PDC-E2, because the removal of anti-PDC-E2 from serum resulted in decreased reactivity to anti-PDC-E2-F(ab)2. Reactivity between PDC-E2 and anti-PDC-E2 from PBC serum was not inhibited by normal sera, indicating that anti-idiotypic antibody from normal sera with anti-PDC-E2 reacts with the framework of F(ab) rather than the paratope. The conclusions are that PBC and normal sera contain IgG class anti-idiotypic antibodies to anti-PDC-E2, the characteristic autoantibody in PBC. Anti-PDC-E2 in immunized animals does not contain an idiotype cross-reactive with human anti-PDC-E2. Anti-idiotypic antibody in PBC is complexed with anti-PDC-E2 and in part accounts for immune complexes demonstrable in PBC. Anti-idiotypic antibody in PBC may regulate levels of anti-PDC-E2.

Adult↗
PubMed1999

Towards the pathogenesis of autoimmune liver disease.

There have been recent improvements in the clinical understanding and definition of the major types of autoimmune liver disease. However, still lacking is knowledge of their prevalence and pathogenesis. Three areas of study are in progress in our laboratory. First, in type 1 autoimmune hepatitis, the search continues to identify a liver/disease-specific autoantigenic reactant. Using hepatocyte membrane preparations, immunoblotting has underlined the problem of distinguishing, among multiple reactants, those that may be causally rather than consequentially related to hepatocellular damage. Second, in primary biliary cirrhosis (PBC), the need for population screening to ascertain prevalence and detect preclinical cases can be met by a rapid automated procedure for detection, by specific enzyme inhibition in microtitre wells, of antibody (anti-M2) to the pyruvate dehydrogenase complex E2 subunit (PDC-E2). Third, the structure of the conformational epitope within the inner lipoyl domain of PDC-E2 is being investigated by screening random phage-displayed peptide libraries using PBC sera. This has yielded phage clones in which the sequence of the peptide insert portrays the structure of this epitope, as judged by clustering of PBC-derived sequences to particular branches of a guide-tree that shows relatedness of peptides, and by reactivity of selected phage clones with anti-PDC-E2. Thus phage display identifies a peptide 'mimotope' of the antibody epitope in the inner lipoyl domain of PDC-E2.

Amino Acid Sequence↗
PubMed1999

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes.

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.

Algorithms↗
PubMed1999

Autoreactive cytotoxic T cells in mice are induced by immunization with a conserved mitochondrial enzyme in Freund's complete adjuvant.

Standard methods to generate autoimmune reactions in mice, by immunization with antigens emulsified with adjuvants, stimulate strong helper (CD4) T-cell and antibody responses but are not reported to induce cytolytic CD8 T cells. The aim of this study was to assess whether specific autoreactive CD8 T cells could be readily generated after immunization with a 'weak' autoantigen in adjuvant. Mice were immunized intraperitoneally three times with the E3 subunit of the mitochondrial 2-oxoacid dehydrogenase enzyme complexes (dihydrolipoamide dehydrogenase) emulsified with Freund's complete adjuvant. Splenic and lymph node lymphocytes were harvested after 14 days for in vitro functional studies. T lymphocytes were tested for proliferative responses and cytotoxicity against antigen-loaded isogeneic target cells. An autoreactive cytolytic T lymphocyte (CTL) response was detectable only after the in vitro restimulation of lymphocytes with E3 antigen-loaded syngeneic splenocytes. These CTL were identified as H-2-restricted CD8+ T cells. A proliferative response to E3 was demonstrable against antigen-pulsed syngeneic splenocytes. Immunized mice also generated strong antibody responses to E3. Liver histology showed portal infiltrates interpreted as a response of the liver to a non-specific immunological stimulus. It is concluded that autoreactive cytolytic T cells can be generated experimentally upon appropriate stimulation of the immune system, and can be identified in vitro upon release from the controlling mechanisms that are likely to regulate them in vivo.

Animals↗
PubMed1999

Phagotopes derived by antibody screening of phage-displayed random peptide libraries vary in immunoreactivity: studies using an exemplary monoclonal antibody, CII-C1, to type II collagen.

Antibody screening of phage-displayed random peptide libraries to identify mimotopes of conformational epitopes is promising. However, because interpretations can be difficult, an exemplary system has been used in the present study to investigate whether variation in the peptide sequences of selected phagotopes corresponded with variation in immunoreactivity. The phagotopes, derived using a well-characterized monoclonal antibody, CII-C1, to a known conformational epitope on type II collagen, C1, were tested by direct and inhibition ELISA for reactivity with CII-C1. A multiple sequence alignment algorithm, PILEUP, was used to sort the peptides expressed by the phagotopes into clusters. A model was prepared of the C1 epitope on type II collagen. The 12 selected phagotopes reacted with CII-C1 by both direct ELISA (titres from < 100-11 200) and inhibition ELISA (20-100% inhibition); the reactivity varied according to the peptide sequence and assay format. The differences in reactivity between the phagotopes were mostly in accord with the alignment, by PILEUP, of the peptide sequences. The finding that the phagotopes functionally mimicked the C1 epitope on collagen was validated in that amino acids RRL at the amino terminal of many of the peptides were topographically demonstrable on the model of the C1 epitope. Notably, one phagotope that expressed the widely divergent peptide C-IAPKRHNSA-C also mimicked the C1 epitope, as judged by reactivity in each of the assays used: these included cross-inhibition of CII-C1 reactivity with each of the other phagotopes and inhibition by a synthetic peptide corresponding to that expressed by the most frequently selected phagotope, RRLPFGSQM. Thus, it has been demonstrated that multiple phage-displayed peptides can mimic the same epitope and that observed immunoreactivity of selected phagotopes with the selecting mAb can depend on the primary sequence of the expressed peptide and also on the assay format used.

Amino Acid Sequence↗
PubMed1999

Rheumatoid arthritis sera react with a phage-displayed peptide selected by a monoclonal antibody to type II collagen that has homology to EBNA-1.

Antibodies to type II collagen, and to Epstein Barr virus nuclear antigen-1 (EBNA-1) have been associated with rheumatoid arthritis (RA). In studies involving probing of phage-displayed random peptide libraries with an antibody to type II collagen, CII-C1, we observed that among 17 phagotopes selected 5 expressed peptides with homology with the sequence of EBNA-1. The residues in common were RLPFG. Hence we tested sera from 50 patients with RA, of whom 26 had antibodies to native type II collagen, and 43 healthy controls, for reactivity by ELISA with a phagotope selected 4 times, which expressed the peptide RRLPFGSQM. Eight RA sera (16%) but no normal sera reacted with the phagotope (p = 0.025). This reactivity could not be correlated with reactivity of RA sera with EBNA-1 by semi-quantitative western blot, with which reactivity occurred in 78% of RA patients and 81% of controls. Evidence for molecular mimicry was not found insofar as the phagotope did not inhibit reactivity of RA sera with EBNA-1 and CII-C1 was not reactive with EBNA-1. We conclude that the reactivity of the RA sera with the phagotope is most likely due to the phagotope being a mimic of an epitope of type II collagen for a proportion of RA sera.

Adult↗
PubMed1998

Expression in Saccharomyces cerevisiae of antigenically and enzymatically active recombinant glutamic acid decarboxylase.

Glutamic acid decarboxylase (GAD) is one of the major autoantigens found in insulin-dependent (Type 1) diabetes mellitus (IDDM). A novel hybrid form of GAD was created by fusing amino acids 1-101 of the human GAD67 protein to amino acids 96-585 of the human GAD65 protein. This hybrid GAD67/65 was expressed constitutively under the control of the phosphoglycerate kinase promoter (PGK1) in the yeast Saccharomyces cerevisiae. Enzymatically active GAD was prepared from yeast lysates by a one-step purification on an affinity column using GAD-1 antibody. The purified hybrid GAD67/65 was radiolabelled with iodine-125 and tested in an immunoprecipitation assay with IDDM sera. Results obtained using the recombinant yeast hybrid GAD67/65 were very similar to those obtained using 125I-labelled porcine GAD. Recombinant yeast hybrid GAD67/65 should have utility for diagnosis and presymptomatic detection of IDDM.

Diabetes Mellitus, Type 1↗
PubMed1998

Autoantibodies to glutamic acid decarboxylase and phenotypic features associated with early insulin treatment in individuals with adult-onset diabetes mellitus.

We investigated the association of serum antibodies to glutamic acid decarboxylase (GADab) with early start of insulin treatment (< or = 1 year from diagnosis, or < or = 2 years from diagnosis) using data from a representative sample of 374 adult-onset insulin-treated individuals from the Tasmanian Diabetes Register. Furthermore, we examined whether this association was stronger than the phenotypic characteristics (age at diagnosis, sex, family history of diabetes, level of obesity, duration of diabetes) often used for diabetes classification. In this cohort, 35.9% of males and 38.5% of females were GADab positive. Within the first year from diagnosis, 78.4% of GADab positive people compared to 44.0% of GADab negative people (p < 0.001) had started insulin treatment. Univariate associations with insulin treatment < or = 1 year from diagnosis included GADab positivity, no family history of diabetes, lower BMI for men, and GADab positivity and lower BMI for women. In multivariate models, significant associations with insulin treatment < or = 1 year from diagnosis included a family history of diabetes (OR = 0.47, 95% CI = 0.23-0.95) and GADab positivity (OR = 2.19, 95% CI = 1.01-4.73) for men, but only GADab positivity (OR = 7.53, 95% CI = 3.09-18.30) for women. Age at diagnosis was not associated with insulin treatment < or = 1 year or < or = 2 years from diagnosis for either sex. These findings indicate that a positive GADab test result is strongly associated with start of insulin treatment within 1 or 2 years from diagnosis, more so than characteristics such as level of obesity and age at diagnosis.

Adult↗
PubMed1998

Natural and disease associated autoantibodies to the autoantigen, dihydrolipoamide acetyltransferase, recognise different epitopes.

Naturally occurring autoantibodies are ubiquitous and may serve physiological functions. We examined the relationship of natural and disease-associated autoantibodies in the context of autoantibodies to dihydrolipoamide acetyltransferase, the 74 kDa E2 sub-unit of the mitochondrial pyruvate dehydrogenase complex (PDC-E2), characteristic of primary biliary cirrhosis (PBC). We tested for natural autoantibodies to PDC-E2 in normal sera, and compared epitopes recognised by natural and disease-associated autoantibodies. Methods included affinity purification of anti-PDC-E2 from normal and PBC sera, ELISA and immunoblotting, capacity of antibodies to inhibit the enzyme function of the pyruvate dehydrogenase complex (PDC), use of F(ab)2 fragments of anti-PDC-E2 in inhibition assays, and testing affinity purified anti-PDC-E2 on peptide fragments of PDC-E2. We found that natural auto-antibodies to PDC-E2 of IgG class were demonstrable in all healthy human sera (10/10). However, their reactivity differed from that of disease-associated autoantibodies, in that anti-PDC-E2 from normal serum failed to inhibit the catalytic activity of PDC; and F(ab)2 fragments from PBC sera potently blocked the binding of anti-PDC-E2 from PBC sera to PDC-E2, but not the binding of natural anti-PDC-E2 to PDC-E2. Immunoblotting on fragments of PDC-E2 using affinity-purified preparations from PBC sera and normal sera failed to provide evidence for gross differences in epitope reactivity. We conclude that normal human sera contain natural IgG autoantibodies to the immunodominant inner lipoyl domain of PDC-E2, as seen characteristically in PBC. However, there is evidence for differences in fine epitope recognition.

Acetyltransferases↗
PubMed1998

Antibodies to ICA512/IA-2 in rodent models of IDDM.

Antibodies to ICA512/IA-2 are a well established marker of human IDDM and can be detected prior to and soon after the onset of insulin dependency. The non-obese diabetic (NOD) mouse and the diabetes-prone BB rat develop spontaneous diabetes as a consequence of T-cell mediated autoimmune destruction of islet beta-cells, but the occurrence of autoantibodies is controversial. We tested sera from NOD mice and BB-rats for anti-ICA512 by a radioimmunoprecipitation assay (RIP). In sequential serum samples from 20 NOD mice, of which 15 developed diabetes, low levels of anti-ICA512 were demonstrable. Anti-ICA512 appeared close to the onset of hyperglycaemia and was usually transient. Non-diabetic NOD mice also produced anti-ICA512, but at a later age and at lower levels than the diabetic NOD mice. In a cross-sectional analysis of sera from BB rats, low levels of anti-ICA512 were present in 11/20 (55%) of non-diabetic-diabetes prone (DP) BB rats, 0/4 (0%) of diabetic DP BB rats, and 1/6 (17%) of diabetes-resistant BB rats. Anti-ICA512 was not detected in rats of other strains, including three Sprague-Dawley rats with streptozotocin-induced diabetes. In both NOD mice and BB rats the anti-ICA512 reactivity was directed to the cytoplasmic domain of the protein. The transient appearance of anti-ICA512 close to the onset of diabetes in NOD mice and the loss of these antibodies after diabetes onset is consistent with the occurrence of anti-ICA512 in human IDDM. Thus in both human IDDM and rodent models, anti-ICA512 is a marker of the impending onset of diabetes and disappears after diabetes onset.

Animals↗
PubMed1998

Mimotopes identified by phage display for the monoclonal antibody CII-C1 to type II collagen.

The characterization of B cell epitopes has been advanced by the use of random peptide libraries displayed within the coat protein of bacteriophage. This technique was applied to the monoclonal antibody (mAb) C1 to type II collagen (CII-C1). CII-C1 is known to react with a conformational epitope on type II collagen that includes residues 359-363. Three rounds of selection were used to screen two random nonameric phage libraries and 18 phagotopes were isolated. CII-C1 reacted by ELISA with 17 of the 18 phagotopes: one phagotope contained a stop codon. Of the eight most reactive phage, seven inhibited the reactivity by ELISA of CII-C1 with type II collagen. Of the 18 phage isolated, 11 encoded the motif F-G-x-Q with the sequence F-G-S-Q in 6, 2 encoded F-G-Q, and one the reverse motif Q-x-y-F. Most phagotopes that inhibited the reactivity of CII-C1 encoded two particular motifs consisting of two basic amino acid residues and a hydrophobic residue in the first part of the insert and the F-G-x-Q or F-G-Q motif in the second part; phagotopes which contained only one basic residue in the first part of the sequence were less reactive. These motifs are not represented in the linear sequence of type II collagen and thus represent mimotopes of the epitope for CII-C1 on type II collagen. There were five phagotopes with peptide inserts containing the sequence RLPFG occurring in the Epstein-Barr virus nuclear antigen, EBNA-1. This is of interest because EBV has been implicated in the initiation of rheumatoid arthritis (RA) by reason of increased reactivity to EBNA-1 in RA sera. In conclusion, the phage display technique disclosed mimotopes for a conformational epitope of type II collagen, and revealed an interesting homology with a sequence of the EBNA-1 antigen from Epstein Barr virus.

Animals↗
PubMed1998

Antibodies to glutamic acid decarboxylase in young Chinese diabetic patients.

Antibodies to glutamic acid decarboxylase (GAD) are a useful autoimmune marker for type 1 diabetes mellitus in Caucasians. We examined antibodies to GAD and their relationships with clinical features and pancreatic beta cell function in 140 young Chinese diabetic patients. Over an 18-month period beginning in 1995, 140 young Chinese diabetic subjects with age of onset of disease < or = 35 years and age < 40 years were recruited consecutively, irrespective of their modes of presentation. Clinical features, antibodies to GAD and pancreatic beta cell function (using a glucagon stimulation test) were examined. Increased levels of antibodies to GAD (> 18 units) were detected in 12.1% (n = 17) of these subjects. Forty-three (31%) patients had a classical type 1 presentation and 65 (46%) patients were insulin-deficient based on post-glucagon plasma C-peptide levels. Patients who were insulin-deficient and had a type 1 presentation had the highest prevalence of antibodies to GAD (29.0%) compared with patients who had a type 2 presentation and were non-insulin deficient (6.4%, P = 0.003). Patients who had antibodies to GAD had lower body mass index and waist-hip ratio, earlier onset of disease, lower blood pressure, plasma triglyceride and C-peptide, and higher concentrations of plasma high-density lipoprotein cholesterol and glycated haemoglobin, and were more likely to require drug treatment, compared with those without antibodies to GAD. In conclusion, there was a low prevalence of antibodies to GAD in Chinese young diabetic patients although such antibodies remained a relatively specific marker for insulin deficiency and acute presentation. Causes other than autoimmunity should be sought to explain the high prevalence of insulin deficiency in these young Chinese patients.

Antibodies↗
PubMed1998

Autoantigenic reactivity of diabetes sera with a hybrid glutamic acid decarboxylase GAD67-65 molecule GAD67(1-101)/GAD65(96-585).

Glutamic acid decarboxylase (GAD) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). Two GAD isoforms exist, GAD65 and GAD67, which differ mostly in the first 100 amino acids of the amino terminus. IDDM sera are predominantly reactive with GAD65 but autoepitopes have been localised only to regions of GAD65 highly homologous with GAD67. In this study we investigated the contribution of the amino terminus to the IDDM epitope on GAD65, in order to test whether this region of GAD could explain the difference in reactivity between GAD65 and GAD67. A recombinant hybrid GAD molecule consisting of amino acids 1-101 of GAD67 and 96-585 of GAD65 was constructed and a truncated GAD65 was also constructed consisting of amino acids 98-585 of GAD65. The reactivity with the hybrid GAD molecule, GAD65 and GAD67, and truncated GAD65 was examined by radioimmunoprecipitation using 50 IDDM sera with known reactivity to purified porcine brain GAD. Over 90% of the IDDM sera were reactive with the hybrid GAD molecule confirming that the amino terminus of GAD65 does not contribute to the autoepitope and that the IDDM epitope is localised to the middle and carboxyl terminal domains of GAD65. Furthermore, evidence is presented that autoantibodies to GAD65 in IDDM sera react with an epitope formed on a dimeric configuration of the molecule.

Autoantigens↗
PubMed1997

Autoantibodies associated with presymptomatic insulin-dependent diabetes mellitus in women.

Presymptomatic autoantibody markers of insulin-dependent (Type 1) diabetes mellitus (IDDM) are less well characterized in adults than in children. We quantitated anti-GAD, anti-ICA512 and ICA by titration to endpoint and compared frequencies and levels in 139 Finnish women from whom 390 serum samples had been archived during antecedent pregnancies for 10 years before and up to 1 year after diagnosis of diabetes. Also, we compared the autoantibody status in adults with IDDM with that of children with newly diagnosed IDDM. Of the 35 women seropositive for 1 or more autoantibodies, 77% developed IDDM, 11% non-insulin-dependent (Type 2) diabetes mellitus (NIDDM), 9% gestational diabetes mellitus requiring insulin (GDM-ins) and 3% GDM controlled by diet. The frequency of antibodies during the 10-year presymptomatic period was 83% for anti-glutamic acid decarboxylase (GAD), 52% for anti-ICA512 and 41% for islet cell antibodies (ICA) for those who developed IDDM, 25%, 17%, and 0% for NIDDM, 12%, 4%, and 8% for GDM-ins and 1%, 0%, and 1% for GDM-diet. Anti-GAD was found most consistently in early samples; 13 of 15 with a single autoantibody at their first test had anti-GAD. Among those who developed IDDM, the frequency of anti-GAD was constant, anti-ICA512 increased threefold, and ICA increased slightly before diagnosis. Levels of the autoantibodies varied between subjects, but were relatively stable in individual subjects. Comparison of tests on the women, and children after diagnosis of IDDM, showed the frequencies and levels to be the same for anti-GAD but lower for anti-ICA512 and ICA in adults. Our observations show in women the long latency of seropositivity before overt IDDM, the predominance of anti-GAD among these three serological markers, and the presence of these markers in NIDDM presumably representing a NIDDM phase of autoimmune insulitis.

Adolescent↗