RESUMO
Objective: Ghrelin is an orexigenic peptide that becomes post-translationally modified. Natural autoantibodies to ghrelin (ghrelin-aAb) have been described in healthy subjects, in eating disorders and rheumatic diseases, with potential clinical relevance. Despite these important reports, the data base on the prevalence and physiological role is small and technical approaches for assessing ghrelin-aAb are few, encouraging respective research for improving knowledge on the potential endocrine significance. Methods: A novel immunoprecipitation assay was generated based on a fusion protein of human ghrelin with a reporter gene. Assay quality was verified with commercial antibodies. Assay characteristics and matrix effects were determined, including stability of natural ghrelin-aAb to freezing, signal linearity in dilution experiments, and comparison of different matrices. Three groups of serum samples were analyzed for ghrelin-aAb, comprising commercial sera from healthy subjects and patients with type 1 or type 2 diabetes mellitus. Results: The newly generated ghrelin-aAb assay proved sensitive, robust and reliable over a broad concentration range. Results from serum and plasma differed slightly. The signals from serum remained stable towards freezing and thawing, and in dilution experiments. Applying a mathematical criterion for outliers (P75 + 1.5-times IQR), an average prevalence of 11%-12% of positive samples was identified in the different human cohorts, with no significant sex-or disease-related difference. General significance: A novel diagnostic autoantibody assay detected ghrelin-aAb with a similar prevalence in diabetic patients and controls, suggesting that autoimmunity to ghrelin plays little role in diabetes mellitus, but may be of relevance in other diseases where ghrelin signaling is essential.
RESUMO
OBJECTIVE: Iodide transport across thyrocytes constitutes a critical step for thyroid hormone biosynthesis, mediated mainly by the basolateral sodium-iodide-symporter (NIS (SLC5A5)) and the apical anion exchanger pendrin (PDS (SLC26A4)). Both transmembrane proteins have been described as autoantigens in thyroid disease, yet the reports on autoantibody (aAb) prevalence and diagnostic usefulness are conflicting. Reasons for the inconclusive findings may be small study groups and principle differences in the technologies used. DESIGN: We decided to re-evaluate this important issue by establishing novel non-radioactive tests using full-length antigens and comparable protocols, and analyzing a large cohort of thyroid patients (n = 323) and control samples (n = 400). METHODS: NIS and PDS were recombinantly expressed as fusion protein with firefly luciferase (Luc). Stably transfected HEK293 cells were used as reproducible source of the autoantigens. RESULTS: Recombinant NIS-Luc showed iodide transport activity, indicating successful expression and correct processing. Commercial antibodies yielded dose-dependent responses in the newly established assays. Reproducibility of assay signals from patient sera was verified with respect to linearity, stability and absence of matrix effects. Prevalence of PDS-aAb was similar in thyroid patients and controls (7.7% vs 5.0%). NIS-aAb were more prevalent in patients than controls (7.7% vs 1.8%), especially in Graves' Disease (12.3%). Neither NIS-aAb nor PDS-aAb concentrations were related to TPO-aAb or TSH-receptor-aAb concentrations, or to serum zinc or selenium status. CONCLUSIONS: Our data highlight a potential relevance of autoimmunity against NIS for thyroid disease, whereas an assessment of PDS-aAb in thyroid patients seems not to be of diagnostic value (yet).