Thyroid Peroxidase as an Autoantigen in Hashimoto's Disease: Structure, Function, and Antigenicity.

Human thyroid peroxidase (TPO), is an important enzyme responsible for the biosynthesis of thyroid hormones and is a major autoantigen in autoimmune thyroid diseases (AITDs) such as the destructive Hashimoto's thyroiditis. Although the structure of TPO has yet to be determined, its extracellular domain consists of three regions that exhibit a high degree of sequence similarity to domains of known three-dimensional structure: the myeloperoxidase (MPO)-like domain, complement control protein (CCP)-like domain, and epidermal growth factor (EGF)-like domain. Homology models of TPO can therefore be constructed, providing some structural context to its known function, as well as facilitating the mapping of regions that are responsible for its autoantigenicity. In this review, we highlight recent progress in this area, in particular how a molecular modelling approach has advanced the visualisation and interpretation of epitope mapping studies for TPO, facilitating the dissection of the interplay between TPO protein structure, function, and autoantigenticity.

Prevalence of pancreatic autoantibodies in non-diabetic patients with autoimmune thyroid disease and its relation to insulin secretion and glucose tolerance

ABSTRACT Objective We evaluated the prevalence of glutamic acid decarboxylase (GADA) and tyrosine phosphatase-protein antibodies (IA2A), their titers and their relation to first phase insulin response (FPIR) and glucose tolerance in autoimmune thyroid diseases (ATDs) patients. Subjects and methods Graves' disease (GD; n = 181) and Hashimoto's thyroiditis (HT; n = 143) patients in addition to healthy controls (n = 93) were studied. Secondly, FPIR and oral glucose tolerance tests (OGTT) were performed in 11 anti-pancreatic islet-cell (+) and in 20 anti-pancreatic-cell (-) patients. Results There was a non significant trend for higher prevalence of GADA positivity in GD vs HT (7.2% vs 2% p = 0.06), but the GADA titers were higher in HT. We also did not find a significant difference in IA2 prevalence (0.7% vs 0.0%) between these two groups or compared to the control group. In the subsequent analysis, low FPIR was found in 10% of these patients but without statistical difference for OGTT between pancreatic antibody-positive and -negative patients. Conclusion A trend for greater prevalence of GADA was observed for GD patients than for HT or control. However, the titers of these autoantibodies were higher in HT patients, but there was no significant relation to insulin secretion and glucose tolerance at that moment and stage of autoimmune diseases.

Prevalence of pancreatic autoantibodies in non-diabetic patients with autoimmune thyroid disease and its relation to insulin secretion and glucose tolerance.

OBJECTIVE: We evaluated the prevalence of glutamic acid decarboxylase (GADA) and tyrosine phosphatase-protein antibodies (IA2A), their titers and their relation to first phase insulin response (FPIR) and glucose tolerance in autoimmune thyroid diseases (ATDs) patients. SUBJECTS AND METHODS: Graves' disease (GD; n = 181) and Hashimoto's thyroiditis (HT; n = 143) patients in addition to healthy controls (n = 93) were studied. Secondly, FPIR and oral glucose tolerance tests (OGTT) were performed in 11 anti-pancreatic islet-cell (+) and in 20 anti-pancreatic-cell (-) patients. RESULTS: There was a non significant trend for higher prevalence of GADA positivity in GD vs HT (7.2% vs 2% p = 0.06), but the GADA titers were higher in HT. We also did not find a significant difference in IA2 prevalence (0.7% vs 0.0%) between these two groups or compared to the control group. In the subsequent analysis, low FPIR was found in 10% of these patients but without statistical difference for OGTT between pancreatic antibody-positive and -negative patients. CONCLUSION: A trend for greater prevalence of GADA was observed for GD patients than for HT or control. However, the titers of these autoantibodies were higher in HT patients, but there was no significant relation to insulin secretion and glucose tolerance at that moment and stage of autoimmune diseases.

Serum anti-Müllerian hormone levels in euthyroid adolescent girls with Hashimoto's thyroiditis: relationship to antioxidant status.

OBJECTIVE: Free radical-mediated oxidative stress has been implicated in the etiopathogenesis of Hashimoto's thyroiditis (HT), which is the most common thyroid disorder in adolescents. HT requires lifelong thyroid surveillance, particularly in women of childbearing age to avoid adverse effects on reproductive function. The aims of this study were to investigate serum concentrations of anti-Müllerian hormone (AMH), a marker of ovarian reserve, in euthyroid adolescent girls with newly diagnosed HT and explore the relationships between AMH levels and biomarkers of antioxidant status. STUDY DESIGN: We recruited 57 non-obese (body mass index [BMI] Z-score<2) adolescent girls with newly diagnosed HT and 50 age- and BMI-matched healthy controls for this case-control study. All participants were euthyroid. Hormonal and metabolic parameters, serum levels of AMH, and antioxidant status [paraoxonase (PON) and arylesterase (ARE) activities] were assessed. RESULTS: Serum AMH levels were significantly higher and serum PON and ARE activities were significantly lower in adolescents with HT than in the controls (p<0.001 for all). No significant associations were detected between the AMH level and any of the clinical or biochemical parameters in the control group. Serum AMH levels were negatively correlated with PON (r=-0.435, p=0.001) and ARE (r=-0.422, p=0.001) activities in adolescents with HT. CONCLUSION: The AMH level was significantly higher while the PON and ARE activities were significantly lower in euthyroid adolescent girls with newly diagnosed HT.

Paraoxonase and arylesterase levels in autoimmune thyroid diseases.

OBJECTIVE: The aim of this study was to evaluate serum paraoxonase-1 (PON1) activity and its association with oxidative stress in autoimmune thyroid disease (AITD). METHODS: A total of 50 patients with AITD, including 25 with Hashimoto's thyroiditis and 25 with Graves' disease were enrolled. The control group comprised 27 healthy subjects. Blood samples were obtained in the euthyroid period and 3 months after initiation of medical treatment. Serum samples from patients with AITD and the healthy control group were analyzed for basal PON1, salt-stimulated PON1, and arylesterase (ARE) activities, along with lipid hydroperoxide (LOOH) and total free sulfhydryl (-SH) levels. RESULTS: Serum PON1 activities and -SH levels were significantly lower (P < 0.001, for each), whereas LOOH levels were significantly higher (P < 0.001, for each) in patients with AITD, compared to the control group. We observed no significant differences in ARE levels between the patient and healthy control groups (P > 0.05). PON1 activity was positively correlated with -SH (r = 0.522, P < 0.001) and negatively correlated with LOOH (r = -0.487, P < 0.001). PON1 phenotype distribution of the subjects was not significantly different among the three groups (P = 0.961). CONCLUSIONS: Serum PON1 activity is decreased in patients with AITD, and correlated positively with -SH, a well-known antioxidant, and negatively with LOOH, an index of lipid oxidation.

Immunoassay of thyroid peroxidase autoantibodies: diagnostic performance in automated third generation methods. A multicentre evaluation.

BACKGROUND: The use of automated immunometric methods for the detection of anti-thyroid peroxidase antibodies (TPOAb), the main serological marker of autoimmune thyroid diseases (AITD), has expanded in recent years. However, it is not known whether these new automated platforms have improved the diagnostic performance of TPOAb assays. The aim of this study was to evaluate the potential improvement of the inter-method agreement of current automated third generation systems, 12 years after a previous study, which had assessed the analytical variability between semi-automated second generation methods of TPOAb detection. METHODS: Eight pools of sera from patients with chronic lymphocytic thyroiditis, exhibiting different TPOAb concentrations, were collected from routine laboratory diagnostics and distributed to seven companies throughout Italy. All automated third generation methods were calibrated against the Medical Research Council (MRC) reference preparation 66/387. RESULTS: The overall mean variability (CV) was 93.6% when results were expressed in part as arbitrary Units (U/mL) and in part as International Units (IU/mL). The conversion of all values in IU/mL resulted in a significant decrease of CV (49.8%). The CV expressed as COM (cut-off concentration multiples) was 64.0%. Agreement of qualitative results was 95.3% with a pronounced difference in the threshold values proposed by manufacturers (range 3.2-35.0 IU/mL). CONCLUSIONS: These findings confirm the improvement of harmonisation between different methods of automated third generation TPOAb assays. Nevertheless, further efforts should be made in the definition of the positive cut-off concentration to avoid misclassification of AITD patients as well as in a new international reference preparation and in the autoantigen purification modality.

Reduced DNASE1 gene expression in thyroid autoimmunity.

The enzyme DNASE1 plays an important role in the hydrolysis of double-stranded DNA and might be related to autoimmunity. Therefore, the authors hypothesized that patients with autoimmune thyroid disease show a reduced expression of the DNASE1 gene. DNASE1 mRNA was quantitatively analyzed in 20 patients (10 with Hashimoto's thyroiditis and 10 with Graves' disease) and 20 age- and sex-matched healthy controls by real-time reverse transcription PCR in a lightcycler using SYBR-Green-format. For relative quantification, the mRNA ratio of the DNASE1 gene to the house keeping gene ß2-MICROGLOBULIN was used. The house keeping gene was proved not to be regulated by autoimmune thyroid disease. The interassay coefficient of variation for patients and controls was 22.2% and 15.6%, respectively, suggesting good reproducibility of measurements. The mean expression of the DNASE1 mRNA in patients was 0.52±0.22 (range 0.18-0.99) and in controls 0.95±0.22 (0.66-1.43). The expression level of the DNASE1 gene was strongly decreased in patients, amounting only 54.7% of that in controls (p<0.001). The lowered expression level in patients was not related to age or sex. This study demonstrated for the first time a downregulation of the DNASE1 mRNA expression in patients with autoimmune thyroid disease. This might result in degrading less DNA from dying cells, thereby promoting the development of thyroid autoimmunity.

Association of polymorphisms in DNMT1, DNMT3A, DNMT3B, MTHFR and MTRR genes with global DNA methylation levels and prognosis of autoimmune thyroid disease.

To clarify the association between factors regulating DNA methylation and the prognosis of autoimmune thyroid diseases (AITDs), we genotyped single nucleotide polymorphisms in genes encoding DNA methyltransferase 1 (DNMT1), DNMT3A, DNMT3B, methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), which are enzymes essential for DNA methylation. Subjects for this study included 125 patients with Hashimoto's disease (HD), including 48 patients with severe HD and 49 patients with mild HD; 176 patients with Graves' disease (GD), including 79 patients with intractable GD and 47 patients with GD in remission; and 83 healthy volunteers (control subjects). The DNMT1+32204GG genotype was more frequent in patients with intractable GD than in patients with GD in remission. Genomic DNA showed significantly lower levels of global methylation in individuals with the DNMT1+32204GG genotype than in those with the AA genotype. The MTRR+66AA genotype was observed to be more frequent in patients with severe HD than in those with mild HD. The DNMT1+14395A/G, DNMT3B-579G/T, MTHFR+677C/T and +1298A/C polymorphisms were not correlated with the development or prognosis of AITD. Our study indicates that the DNMT1+32204GG genotype correlates with DNA hypomethylation and with the intractability of GD, and that the MTRR+66AA genotype may correlate with the severity of HD.

Association between TPO gene polymorphisms and Anti-TPO level in Tehranian population: TLGS.

INTRODUCTION: Thyroid peroxidase (TPO) gene variations are one cause of thyroid autoimmune diseases. The aim of this study was to examine the association between the T1936C, T2229C and A2257C polymorphisms of the TPO gene and Anti-TPO level. MATERIALS AND METHODS: In this case-control study, 188 individuals (86 males and 102 females), aged 20-80 years, were randomly selected from among the Tehran Lipid and Glucose Study (TLGS) population. A2257C and T2229C SNPs were detected with RFLP by use of BsrI and Eco57I as the restriction enzymes respectively, while the T1936C SNP was determined with ARMS-PCR. RESULTS: In the presence of the C allele of T1936C, Anti-TPO level was significantly increased (CC: 238±43.3, CT: 47.7±15.9, TT: 74.1±11.3 IU/L p=0.002); however, this association was attenuated after adjustment for sex and age (p=0.059). No significant difference, before and after adjustment, was found in Anti-TPO level in the presence of T2229C SNP (CC: 129.1±24.5, CT: 43.5±12.6, TT: 126.5±13.8 IU/L p=0.196). The association between A2257C and Anti-TPO level was only significant after adjustment for potential confounders (p=0.007). The association between ATC and CTT haplotypes and Anti-TPO level was significant (p=0.023, 0.021 respectively), the association between CTT and Anti-TPO concentration was also significant after adjustment for sex (p=0.014). CONCLUSION: The results of the present study confirmed the association between TPO gene polymorphisms and Anti-TPO level in the Tehranian population.

Protein kinase Cε regulates proliferation and cell sensitivity to TGF-1ß of CD4+ T lymphocytes: implications for Hashimoto thyroiditis.

We have studied the functional role of protein kinase Cε (PKCε) in the control of human CD4(+) T cell proliferation and in their response to TGF-1ß. We demonstrate that PKCε sustains CD4(+) T cell proliferation triggered in vitro by CD3 stimulation. Transient knockdown of PKCε expression decreases IL-2R chain transcription, and consequently cell surface expression levels of CD25. PKCε silencing in CD4 T cells potentiates the inhibitory effects of TGF-1ß, whereas in contrast, the forced expression of PKCε virtually abrogates the inhibitory effects of TGF-1ß. Being that PKCε is therefore implicated in the response of CD4 T cells to both CD3-mediated proliferative stimuli and TGF-1ß antiproliferative signals, we studied it in Hashimoto thyroiditis (HT), a pathology characterized by abnormal lymphocyte proliferation and activation. When we analyzed CD4 T cells from HT patients, we found a significant increase of PKCε expression, accounting for their enhanced survival, proliferation, and decreased sensitivity to TGF-1ß. The increased expression of PKCε in CD4(+) T cells of HT patients, which is described for the first time, to our knowledge, in this article, viewed in the perspective of the physiological role of PKCε in normal Th lymphocytes, adds knowledge to the molecular pathophysiology of HT and creates potentially new pharmacological targets for the therapy of this disease.

[Cytofluorometric analysis of chosen markers of apoptosis CD95/CD95L (Fas/FasL) in thyroid tissues from young patients with Graves' disease and Hashimoto's thyroiditis]. / Cytofluorymetryczna analiza wybranych markerów apoptozy Fas/FasL (CD95/CD95L) w tkance gruczolu tarczowego u mlodocianych pacjentów z choroba Gravesa-Basedowa oraz zapaleniem tarczycy typu Hashimoto.

BACKGROUND: Apoptosis, one of the forms of programmed cell death, is a physiologic process of cell death that is central to normal development and occurs in response to a variety of physiologic and pathophysiologic stimuli. In the thyroid, abnormal apoptotic activity may be involved in a variety of diseases such as Hashimoto thyroiditis and Graves disease. The aim of this study was to estimate the expression of chosen apoptotic molecules CD95 (Fas) and CD95L (FasL) on the surface of thyroid follicular cells in application of mouse monoclonal antibodies #64 which recognized B antigen regions of thyroid peroxidase (TPO) and infiltrating inflammatory cells. MATERIAL AND METHODS: The investigation was performed on thyroid cells isolated from surgically treated thyroid tissues of 15 patients with Graves' disease (GD), 15 patients with a nontoxic multinodular goiter (NTMG) and 15 aspirates obtained by FNAB from patients with Hashimoto thyroiditis (HT). The thyrocytes were identified by an indirect method: in the first stage we added mouse monoclonal autoantibodies specific for TPO (mAb #64) regions and in the second stage we conjugated this complex with rabbit anti-mouse antibodies IgG (Fab')2 with FITC. In the next step the cellular suspension was completed with suitably well-chosen two-colour monoclonal antibodies marked (PE or PerCP) (Becton Dickinson) directed against suitable apoptotic (Fas/FasL) molecules. All investigations were performed by flow cytometry using Coulter EPICS XL apparatus. RESULTS: The percentages of thyroid cells were estimated with expression of region B antigenic TPO in reference to individual apoptotic molecules. The analysis of Fas and FasL expression in thyroid tissues revealed significantly increased percentage of intrathyroidal T cells with CD95+ (p<0.005, p<0.001), CD95L+ (p<0.02, p<0.01) and both CD95/CD95L (ns, p<0.05) expression in comparison to percentages of T cells in patients with HT and NTMG. In addition, on the surface of thyroid follicular cells in patients with GD (p<0.01, p<0.01) and NTMG (p<0.001, p<0.004) we observed a lower percentage of thyrocytes with CD95 and CD95L molecules than in cases with HT. The expression of both apoptotic molecules on thyroid cells was higher (18%) in patients with HT in comparison to the percentages of positive cells in patients with GD (p<0.02, p<0.002) and NTMG, 8% and 1%, respectively. CONCLUSIONS: We conclude that alterations in the expression of death receptors and their ligands on the surface of thyroid follicular cells may play a role in the regulation of apoptosis in thyroid autoimmune disorders.