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Aims: Glutamate decarboxylase (GAD) antibodies are the most widely used predictive marker for Type 1 diabetes, but many individuals currently found to be GAD antibody-positive are unlikely to develop diabetes. We have shown previously that radioimmunoassays using N-terminally truncated 35 S-GAD 65 (96-585) offer better disease specificity with similar sensitivity to full-length 35 S-GAD 65 (1-585). To determine whether assay performance could be improved further, we evaluated a more radically truncated 35 S-GAD 65 (143-585) radiolabel., Methods: Samples from people with recent-onset Type 1 diabetes (n = 157) and their first-degree relatives (n = 745) from the Bart's-Oxford family study of childhood diabetes were measured for GAD antibodies using 35 S-labelled GAD 65 (143-585). These were screened previously using a local radioimmunoassay with 35 S-GAD 65 (1-585). A subset was also tested by enzyme-linked immunosorbent assay (ELISA), which performs well in international workshops, but requires 10 times more serum. Results were compared with GAD antibody measurements using 35 S-GAD 65 (1-585) and 35 S-GAD 65 (96-585)., Results: Sensitivity of GAD antibody measurement was maintained using 35 S-GAD 65 (143-585) compared with 35 S-GAD 65 (1-585) and 35 S-GAD 65 (96-585). Specificity for Type 1 diabetes was improved compared with 35 S-GAD 65 (1-585), but was similar to 35 S-GAD 65 (96-585). Relatives found to be GAD antibody-positive using these truncated labels were at increased risk of diabetes progression within 15 years, compared with those positive for GAD(1-585) antibody only, and at similar risk to those found GAD antibody-positive by ELISA., Conclusions: The first 142 amino acids of GAD 65 do not contribute to epitopes recognized by Type 1 diabetes-associated GAD antibodies. Low-volume radioimmunoassays using N-terminally truncated 35 S-GAD 65 are more specific than those using full-length GAD 65 and offer practical alternatives to the GAD antibody ELISA for identifying children at increased risk of Type 1 diabetes., (© 2018 Diabetes UK.)

The synthesis of arginine vasopressin (AVP) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus is sensitive to increased plasma osmolality and a decreased blood volume, and thus is robustly increased by both dehydration (increased plasma osmolality and decreased blood volume) and salt loading (increased plasma osmolality). Both stimuli result in functional remodelling of the SON and PVN, a process referred to as functional-related plasticity. Such plastic changes in the brain have recently been associated with altered patterns of DNA methylation at CpG (cytosine-phosphate-guanine) residues, a process considered to be important for the regulation of gene transcription. In this regard, the proximal Avp promoter contains a number of CpG sites and is recognised as one of four CpG islands for the Avp gene, suggesting that methylation may be regulating Avp transcription. In the present study, we show that, in an immortalised hypothalamic cell line 4B, the proximal Avp promoter is highly methylated, and treatment of these cells with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine to demethylate DNA dramatically increases basal and stimulated Avp biosynthesis. We report no changes in the expression of DNA methyltransferases, Dnmt1 and Dnmt3a, whereas there is decreased expression of the demethylating enzyme ten-eleven-translocation 2, Tet2, in the SON by dehydration and salt loading. We found higher methylation of the SON Avp promoter in dehydrated but not salt-loaded rats. By analysis of individual CpG sites, we observed hypomethylation, hypermethylation and no change in methylation of specific CpGs in the SON Avp promoter of the dehydrated rat. Using reporter gene assays, we show that mutation of individual CpGs can result in altered Avp promoter activity. We propose that methylation of the SON Avp promoter is necessary to co-ordinate the duel inputs of increased plasma osmolality and decreased blood volume on Avp transcription in the chronically dehydrated rat., (© 2016 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.)