Thyroid peroxidase forms thionamide-sensitive homodimers: relevance for immunomodulation of thyroid autoimmunity

Thyroid peroxidase (TPO) is the key enzyme in thyroid hormone production and a universal autoantigen in Graves’ and other autoimmune thyroid diseases. We wished to explore the expression of TPO and whether it was affected by thionamide antithyroid drugs. We studied recombinant TPO, stably expressed by a Chinese hamster ovary cell line (CHO-TPO) and transiently expressed TPO-enhanced green fluorescent protein (eGFP) and -FLAG fusion proteins. Immunoblotting of CHO-TPO cell extracts showed high-molecular weight (HMW) TPO isoforms that were resistant to reduction, as well as 110 kDa monomeric TPO. Co-immunoprecipitation and enzyme-linked-immunosorbent assay (ELISA) binding studies of FLAG- and eGFP-tagged TPO demonstrated TPO dimerisation. CHO-TPO cells cultured in methimazole (MMI) for 10 days showed a significant reduction in HMW-TPO isoforms at MMI concentrations of 1 µM and above (p < 0.01), whereas monomeric TPO expression was unchanged. We observed a similar reduction in HMW-TPO in CHO-TPO cells cultured in propylthiouracil (10 µM and above). Binding of Graves’ disease patient sera and TPO-Fabs to enzymatically active TPO that was captured onto solid phase was not abrogated by MMI. The cellular localisation of TPO in CHO-TPO cells was unchanged by MMI treatment. Our demonstration of homodimeric TPO and the reduction in HMW-TPO isoforms during thionamide treatment of CHO-TPO cells shows, for the first time, an effect of thionamides on TPO structure. This suggests a structural correlate to the effect of thionamides on TPO enzymatic activity and opens up a novel potential mechanism for thionamide immunomodulation of autoimmune thyroid disease

Different fetal-neonatal outcomes in siblings born to a mother with Graves-Basedow disease after total thyroidectomy: a case series

ABSTRACT:INTRODUCTION: We describe three different fetal or neonatal outcomes in the offspring of a mother who had persistent circulating thyrotropin receptor antibodies despite having undergone a total thyroidectomy several years before. CASE PRESENTATION: The three different outcomes were an intrauterine death, a mild and transient fetal and neonatal hyperthyroidism and a severe fetal and neonatal hyperthyroidism that required specific therapy. CONCLUSIONS: The three cases are interesting because of the different outcomes, the absence of a direct correlation between thyrotropin receptor antibody levels and clinical signs, and the persistence of thyrotropin receptor antibodies several years after a total thyroidectomy

Comprehensive Genotyping in Two Homogeneous Graves' Disease Samples Reveals Major and Novel HLA Association Alleles

BACKGROUND: Graves' disease (GD) is the leading cause of hyperthyroidism and thyroid eye disease inherited as a complex trait. Although geoepidemiology studies showed relatively higher prevalence of GD in Asians than in Caucasians, previous genetic studies were contradictory concerning whether and/or which human leukocyte antigen (HLA) alleles are associated with GD in Asians. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a case-control association study (499 unrelated GD cases and 504 controls) and a replication in an independent family sample (419 GD individuals and their 282 relatives in 165 families). To minimize genetic and phenotypic heterogeneity, we included only ethnic Chinese Han population in Taiwan and excluded subjects with hypothyroidism. We performed direct and comprehensive genotyping of six classical HLA loci (HLA-A, -B, -C, -DPB1, -DQB1 and -DRB1) to 4-digit resolution. Combining the data of two sample populations, we found that B*46:01 (odds ratio under dominant model [OR]  = 1.33, Bonferroni corrected combined P [P(Bc)]  = 1.17 x 10⁻²), DPB1*05:01 (OR  = 2.34, P(Bc) = 2.58 x 10⁻¹⁰), DQB1*03:02 (OR  = 0.62, P(Bc)  = 1.97 x 10⁻²), DRB1*15:01 (OR  = 1.68, P(Bc) = 1.22 x 10⁻²) and DRB1*16:02 (OR  = 2.63, P(Bc)  = 1.46 x 10⁻⁵) were associated with GD. HLA-DPB1*05:01 is the major gene of GD in our population and singly accounts for 48.4% of population-attributable risk. CONCLUSIONS/SIGNIFICANCE: These GD-associated alleles we identified in ethnic Chinese Hans, and those identified in other Asian studies, are totally distinct from the known associated alleles in Caucasians. Identification of population-specific association alleles is the critical first step for individualized medicine. Furthermore, comparison between different susceptibility/protective alleles across populations could facilitate generation of novel hypothesis about GD pathophysiology and indicate a new direction for future investigation

Thyroid function, autoimmunity and nodules in hematological malignancies

Objective Hematological malignancies encompass a large spectrum of disease entities whose treatment by chemo/radiotherapy could lead to thyroid complications. To the best of our knowledge, no study has simultaneously addressed thyroid function, autoimmunity and nodularity. Therefore, we decided to conduct one.Materials and methods We evaluated 82 Caucasian patients (36 women and 46 men), who were treated at our Oncology division for hematological malignancies (multiple myeloma, chronic myeloid leukemia, chronic lymphatic leukemia, non-Hodgkin lymphoma and polycythemia vera) and compared them with a control group of 104 patients. Patients who had received or were receiving external head/neck radiotherapy were excluded. All oncological patients and control individuals underwent thyroid ultrasonography and thyroid function and autoimmunity tests.Results A lower prevalence of enlarged thyroid and nodules were found in patients with respect to controls. The rate of thyroid nodules was the highest in multiple myeloma and polycythemia vera, and the lowest in chronic lymphatic leukemia. Non-Hodgkin lymphoma patients had the smallest thyroid nodules while men with multiple myeloma the biggest ones. No patient had hypothyroidism, while 5.6% of patients had subclinical hyperthyroidism. In contrast, within the control group the rates of hypothyroidism and hyperthyroidism, overt and subclinical, were 3.8%, 20.2%, 0% and 0% respectively. Moreover, the overall rate of thyroid autoantibody positiveness in patients was significantly lower than controls.Conclusion In our experience, we found a significantly lower prevalence of thyroid abnormalities in hematologic patients who underwent chemotherapy, but not radiotherapy, with respect to controls. Arch Endocrinol Metab. 2015;59(3):236-4

Graves' disease is associated with a defective expression of the immune regulatory molecule galectin-9 in antigen-presenting dendritic cells

Introduction Patients with autoimmune thyroid disease (AITD) show defects in their immune-regulatory mechanisms. Herein we assessed the expression and function of galectin-1 and galectin-9 (Gal-1, Gal-9) in dendritic cells (DCs) from patients with AITD. Materials and Methods Peripheral blood samples from 25 patients with Graves’ disease (GD), 11 Hashimoto’s thyroiditis (HT), and 24 healthy subjects were studied. Thyroid tissue samples from 44 patients with AITD and 22 patients with goiter were also analyzed. Expression and function of Gal-1 and Gal-9 was assessed by quantitative RT-PCR, immunofluorescence and flow cytometry. Results A diminished expression of Gal-9, but not of Gal-1, by peripheral blood DCs was observed in GD patients, mainly in those with Graves´ ophthalmopathy, and a significant negative association between disease severity and Gal-9 expression was detected. In addition, the mRNA levels of Gal-9 and its ligand TIM-3 were increased in thyroid tissue from AITD patients and its expression was associated with the levels of Th1/Th12/Th17 cytokines. Immunofluorescence studies proved that intrathyroidal Gal-9 expression was confined to DCs and macrophages. Finally, in vitro functional assays showed that exogenous Gal-9 had a suppressive effect on the release of Th1/Th2/Th17 cytokines by DC/lymphocyte autologous co-cultures from both AITD patients and healthy controls. Conclusions The altered pattern of expression of Gal-9 in peripheral blood DCs from GD patients, its correlation with disease severity as well as its ability to suppress cytokine release suggest that Gal-9 could be involved in the pathogenesis of AITDThis work was supported by grants from the Fondo de Investigaciones Sanitarias (FISS) PI10/ 02521 and S2010/BMD-2328 TIRONET (Comunidad de Madrid), Spain (to MM) and the Fondo de Cooperación Internacional en Ciencia y Tecnología (FONCICYT) 95395, European Union-México (to RGA