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.

Electrochemical sensor for blood deoxyribonucleases: design and application to the diagnosis of autoimmune thyroiditis.

We designed an electrochemical sensor based on a carbon nanotube modified electrode (ME) to analyze DNA-cleaving activity. The cleavage of high molecular weight DNA resulted in an increase in the oxidation current from DNA guanine nucleotides due to a change in DNA adsorptive behavior on the surface of the ME. DNA digestion with DNAse I was accompanied by a linear increase in the DNA signal in proportion to the enzyme activity. We then proposed an assay based on the sensor for the direct assessment of the total deoxyribonuclease activity of blood serum as well as the separate detection of DNAse I and DNA abzymes. The assay was applied to analyze deoxyribonucleases in sera from 21 healthy donors and 17 patients with autoimmune thyroiditis. Our results show that the response of the sensor to DNA cleavage by blood deoxyribonucleases is a promising diagnostic criterion for autoimmune thyroiditis. This sensor can be implemented in a disposable screen-printed electrode format for application in clinical laboratories.

Structural Studies of Thyroid Peroxidase Show the Monomer Interacting With Autoantibodies in Thyroid Autoimmune Disease.

Thyroid peroxidase (TPO) is a critical membrane-bound enzyme involved in the biosynthesis of multiple thyroid hormones, and is a major autoantigen in autoimmune thyroid diseases such as destructive (Hashimoto) thyroiditis. Here we report the biophysical and structural characterization of a novel TPO construct containing only the ectodomain of TPO and lacking the propeptide. The construct was enzymatically active and able to bind the patient-derived TR1.9 autoantibody. Analytical ultracentrifugation data suggest that TPO can exist as both a monomer and a dimer. Combined with negative stain electron microscopy and molecular dynamics simulations, these data show that the TR1.9 autoantibody preferentially binds the TPO monomer, revealing conformational changes that bring together previously disparate residues into a continuous epitope. In addition to providing plausible structural models of a TPO-autoantibody complex, this study provides validated TPO constructs that will facilitate further characterization, and advances our understanding of the structural, functional, and antigenic characteristics of TPO, an autoantigen implicated in some of the most common autoimmune diseases.

Prunella vulgaris L. attenuates experimental autoimmune thyroiditis by inducing indoleamine 2,3-dioxygenase 1 expression and regulatory T cell expansion.

BACKGROUND: Prunella vulgaris L. (P. vulgaris) has traditionally been used to treat swelling and inflammation of the thyroid gland. This study aimed to evaluate the effects of P. vulgaris on experimental autoimmune thyroiditis (EAT) and explore the roles of indoleamine 2,3-dioxygenase 1 (IDO1) and regulatory T cells (Tregs) in these P. vulgaris-mediated effects. METHODS: The main bioactive compounds in P. vulgaris were analysed by high-performance liquid chromatography. An EAT model was established by immunization of Lewis rats with thyroglobulin via subcutaneous injection. Thyroid volume was assessed by ultrasound, and lymphatic infiltration in the thyroid was evaluated by haematoxylin and eosin staining. The serum levels of thyroglobulin antibody (TgAb) and cytokines were measured by indirect enzyme-linked immunosorbent assay. The percentage of CD4+CD25+Foxp3+ Tregs was detected by flow cytometry. The mRNA and protein levels of IDO1 were measured by qRT-PCR and Western blotting, respectively. The levels of tryptophan (Trp) and kynurenine (Kyn) in serum and faecal samples were assessed with a fluorometric kit and spectrophotometry. RESULTS: The main bioactive compound in P. vulgaris was rosmarinic acid. The TgAb level and thyroid volume in EAT rats were significantly decreased after administration of P. vulgaris (P < 0.01). The inflammation score in EAT rats that were administered P. vulgaris was significantly lower than that in the EAT controls (P < 0.01). In addition, P. vulgaris promoted the expansion of splenic Tregs and increased the production of IL-10 and TGF-ß (P < 0.01) in EAT rats. Moreover, P. vulgaris induced IDO1 mRNA and protein expression in the spleen and intestine in P. vulgaris-treated EAT rats (P < 0.01). Finally, Trp levels were reduced and Kyn levels and the Kyn/Trp ratio were increased in the serum of P. vulgaris-treated EAT rats. CONCLUSION: We were the first to demonstrate the role of IDO1-induced Treg expansion in P. vulgaris-mediated attenuation of EAT. Our study provides insight into the immunopathogenesis of autoimmune thyroiditis and shows the potential therapeutic value of P. vulgaris.

Thyroid autoimmunity in patients with alopecia areata.

Alopecia areata (AA) is a common form of localized, non-scarring hair loss. It is characterized by the loss of hair in patches, total loss of scalp hair (alopecia totalis), or total loss of body hair (alopecia universalis). The etiopathogenesis of the disease is still unclear, but there is evidence that autoimmunity and endocrine dysfunction may be involved. The aim of this study was to determine whether AA is statistically associated with thyroid autoimmunity. In this retrospective epidemiologic study, we compared the frequency of thyroid autoantibodies (thyroglobulin antibody, TgAb, and thyroid peroxidase antibody, TPAb) ATPO) in 70 AA patients and 30 healthy volunteers. Thyroid autoantibodies and thyroid hormones (thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH)) were measured in all subjects. Thyroid functional abnormalities were found in 8 (11.4%) AA patients. Positive autoimmune antibodies were associated with AA in 18 (25.7%) patients, with no significant association between the disease severity and presence of these antibodies. The frequency of thyroid autoantibodies was significantly higher in AA patients than in healthy controls (25.7% vs. 3.3%; p<0.05). Our findings pointed to a significant association between AA and thyroid autoimmunity and showed the tests to detect thyroid autoantibodies to be relevant in AA patients.

Impaired caspase-3 expression by peripheral T cells in chronic autoimmune thyroiditis and in autoimmune polyendocrine syndrome-2.

CONTEXT: Activation-induced cell death (AICD) is a major mechanism in the regulation of peripheral tolerance, and caspase-3 represents its major executioner. AICD impairment contributes to the persistence of autoreactive T cells, and defective AICD has been reported in autoimmune thyroiditis as well as in type 1 diabetes mellitus. OBJECTIVE: The objective of this study was to evaluate the involvement of caspase-3 in the regulation of AICD resistance in thyroid and polyendocrine autoimmunity. DESIGN/SETTINGS/PATIENTS/INTERVENTION: Caspase-3 expression was analyzed in peripheral blood lymphocytes from 26 adults (A-AT) and 25 children (Y-AT) affected by autoimmune thyroiditis and 13 individuals affected by chronic autoimmune thyroiditis plus Addison's disease [autoimmune polyendocrine syndrome-2 (APS-2)] in comparison with 32 age-matched normal control subjects (NC). OUTCOME MEASURES: Caspase-3 mRNA expression in peripheral T cells was evaluated by quantitative real-time PCR; protein expression of both procaspase-3 and activated caspase-3 by Western blot analysis was followed by scanning densitometry. RESULTS: Caspase-3 mRNA expression was significantly reduced in resting lymphocytes from both A-AT (P = 0.001) and Y-AT (P = 0.016) compared with NC. After lymphocyte activation, protein levels of caspase-3 active form were significantly reduced in A-AT (P = 0.023) and Y-AT (P = 0.001) compared with NC. The APS-2 group displayed characteristics similar to the A-AT group because both caspase-3 mRNA and protein active form levels were significantly reduced compared with NC (P = 0.004 and 0.002, respectively). CONCLUSION: Our data show that peripheral lymphocytes of subjects affected by thyroid autoimmunity or APS-2 show defective expression of the major executioner of AICD, thus potentially contributing to AICD resistance and to the development of autoimmunity.

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.

Thyroglobulin and thyroid peroxidase share common epitopes recognized by autoantibodies in patients with chronic autoimmune thyroiditis.

Monoclonal antibodies specific for human thyroid peroxidase (TPO) were prepared by the hybridoma technique using hyperimmune spleen cells from mice immunized with TPO purified from thyroid glands from patients with Graves' disease. Use of the microenzyme-linked immunosorbent assay method revealed that some of the monoclonal antibodies cross-reacted strongly with human thyroglobulin (Tg). Conversely, monoclonal anti-Tg antibodies cross-reacted with TPO, albeit to a lesser degree. Some anti-Tg autoantibodies in serum from patients with chronic autoimmune thyroiditis purified by Tg affinity chromatography bound TPO, and such binding was completely inhibited by Tg. Western blotting experiments revealed that thyroid microsomal 103K proteins recognized by mouse monoclonal and polyclonal anti-TPO antibodies were recognized by some monoclonal anti-Tg antibodies and anti-Tg autoantibodies, and conversely, that 19S Tg was recognized by some monoclonal anti-TPO antibodies. TPO was immunoprecipitated by anti-Tg autoantibodies isolated by Tg affinity chromatography. On the other hand, the specificity for TPO of the anti-Tg autoantibodies was not identical with that of anti-TPO autoantibodies. These cross-reactivities were not due to contamination of TPO with Tg or vice versa, or to contamination of the anti-Tg autoantibody preparations with anti-TPO autoantibodies. Taken together, these data indicate that Tg and TPO share common antigenic determinants and that some of those determinants are recognized by autoantibodies in the serum of patients with chronic autoimmune thyroiditis.

Deficiency of superoxide dismutase in endemic goiter tissue.

Superoxide dismutase (SOD) activity and its concentration were measured in thyroid tissues obtained from patients with Graves' disease, Hashimoto's thyroiditis, differentiated thyroid cancer, and endemic goiter (before and after iodine supplementation) as well as in normal thyroid tissue (paranodular tissue) from patients with follicular adenomas. SOD activity was measured by pyrogallol assay in ethanol-chloroform extracts of the thyroid homogenates. The SOD concentration in the thyroid extract was measured as immunoreactive SOD by electroimmunoassay. Endemic goiter tissues (n = 10) contained significantly lower SOD activity [mean, 1.9 +/- 1.9 (+/- SD) vs. 7.5 +/- 3.9 ng purified SOD/micrograms DNA; P less than 0.02] and concentration (mean, 0.2 +/- 0.1 vs. 0.8 +/- 0.5 ng SOD/microgram DNA; P less than 0.01) compared with those of normal tissues. No other pathological thyroid tissues had such consistently low SOD levels. Lactate dehydrogenase activity, a marker of cytosolic enzyme, was not lower in endemic goiter tissues than in normal tissues, suggesting that both tissues possessed functioning cells capable of producing cytosolic enzyme. Thyroid tissue from endemic goiter patients previously treated with iodized oil injection also had low SOD activity and concentration. Western blot analysis indicated that SOD protein in the endemic goiter tissue did not differ from that in normal thyroid tissue. We conclude that there is deficiency of cytosolic SOD in endemic goiter tissue. Since the deficiency of cytosolic SOD causes more prolonged exposure to oxygen free radicals, the decrease in SOD might contribute to the degenerative changes frequently found in these tissues.

Recombinant human thyroid peroxidase generated in eukaryotic cells: a source of specific antigen for the immunological assay of antimicrosomal antibodies in the sera of patients with autoimmune thyroid disease.

Recombinant, enzymatically active human thyroid peroxidase (hTPO) generated in nonthyroidal eukaryotic cells was compared with Graves' thyroid microsomes as a source of antigen for the immunological detection of antimicrosomal/anti-hTPO antibodies. Enzyme-linked immunosorbent assay of 51 sera, selected to produce a balanced distribution of antimicrosomal antibody (anti-MSA) levels, revealed (at 1:100 serum dilution) a moderately good correlation between anti-MSA and anti-hTPO antibody levels (r = 0.668; P less than 0.001). However, a number of sera with high anti-MSA levels yielded markedly discordant values between the two assays. A much lower correlation was observed between antithyroglobulin and anti-hTPO antibody levels (r = 0.315; P less than 0.05). At higher serum dilutions (1:1,000 and 1:10,000), at which low affinity, high capacity binding reactions will be reduced, the correlation between anti-MSA and anti-hTPO antibody values was greatly improved (r = 0.906 and 0.902, respectively; P less than 0.001), and there were no longer widely discrepant values between the two assays. In summary, the present study indicates that recombinant hTPO expressed in nonthyroidal cells provides an unlimited source of human TPO of unvarying quality for anti-hTPO antibody assays. This material offers increased specificity over standard anti-MSA assays that use thyroid cell microsomes as antigen.

Thyroid peroxidase activity-inhibiting immunoglobulins in patients with autoimmune thyroid disease.

The thyroid microsomal antibody (M-Ab) has been found to be an antibody against thyroid peroxidase (TPO), and such antibodies have been reported not only to bind TPO but also to directly inhibit TPO activity. In this study we investigated the relationship between TPO activity-inhibiting immunoglobulin (TPII) and thyroid function in 55 untreated patients with hyperthyroidism due to Graves' disease and 35 untreated patients with Hashimoto's disease. TPO partially purified from the microsomal fraction of Graves' thyroid tissue by Sephacryl S-300 gel filtration was incubated with immunoglobulin (Ig) fractions of serum prepared by precipitation with 15% polyethylene glycol. At the end of incubation, TPO activity was measured by a guaiacol assay. The TPII level was expressed as the TPII index, defined as the inhibition of TPO activity by patient Ig divided by inhibition produced by a known positive Ig. We also measured serum free T4, free T3, and TSH concentrations and anti-M-Ab titers, the latter by a microenzyme-linked immunosorbent assay. When a positive TPII index was defined as more than the mean + 2 SD of the TPII index (0.38) for 15 normal subjects, 13 patients with Graves' disease and 14 patients with Hashimoto's disease had positive TPII index values. There was a positive correlation between the TPII index values and the M-Ab titers in patients with either Graves' disease (r = 0.38; P less than 0.01) or Hashimoto's disease (r = 0.52; P less than 0.01). The mean TPII index in patients with Hashimoto's disease was significantly higher than that in patients with Graves' disease [0.38 +/- 0.42 (+/- SD) vs. 0.19 +/- 0.41; P less than 0.05]. The slope of the regression line between the TPII index values and the M-Ab titers for patients with Hashimoto's disease was steeper than that for patients with Graves' disease. The mean serum free T4 concentration was significantly lower in those patients with Hashimoto's disease who had positive TPII index values than in those with negative TPII index values (14.0 +/- 5.0 vs. 9.6 +/- 3.7 pmol/L; P less than 0.01). There was no significant difference in thyroid function between the patients with Graves' disease with positive and negative TPII index values. TPII appears to inhibit thyroid function in some patients, but no simple relationship between TPII and thyroid function in autoimmune thyroid disease was demonstrated. Understanding the factors that control access of anti-TPO antibody to its antigen may help to elucidate the significance of circulating anti-TPO antibody.

Identification of localized autoantibody epitopes in thyroid peroxidase.

Recent reports have disagreed on the nature of the autoantibody epitopes in thyroid peroxidase (TPO). We used immunoprecipitation of recombinant human TPO constructs to determine if localized autoantibody binding sites exist in this autoantigen. In vitro transcription and translation of TPO cDNA fragments yielded 35S-labeled products consisting of either full-length protein (933 amino acids) or N-terminal peptides of 631, 455, and 120 amino acids. Immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the Hashimoto's sera consistently precipitated the full-length and the 631 amino acid products, but not the shorter N-terminal peptides. An additional construct resulting in a full-length TPO peptide with an internal deletion of amino acids 4-455 was also made, and this product was also precipitated by the Hashimoto's sera. A fusion protein consisting of maltose binding protein followed by amino acids 456-933 of human TPO was produced in Escherichia coli and subjected to Western blot analysis using the Hashimoto's sera. The Hashimoto's sera reacted with the MalTose binding protein TPO (MBP/TPO) fusion protein, but not a control fusion protein (MBP/LacZ alpha). Together, these results indicate the presence of localized autoantibody epitopes in the portion of the human TPO molecule from amino acids 456 to 933, with at least one binding site located between amino acids 456 and 631.

Autosomal dominant transmission of autoantibodies to thyroglobulin and thyroid peroxidase.

The inheritance of autoantibodies to thyroglobulin and thyroid peroxidase (thyroid microsomal antigen) has been reevaluated with newly developed ultrasensitive assays that depend on the direct interaction between antibody and radiolabeled antigen. In a study of 16 families with autoimmune thyroid disease, autoantibodies to thyroid peroxidase (TPO) were found to be inherited as a dominant Mendelian trait in females with reduced penetrance in males. Similar results were obtained with thyroglobulin (Tg) autoantibodies. Genetic linkage analysis of the loci for TPO and Tg autoantibodies with 28 polymorphic serological markers (including HLA and Gm allotypes) was carried out in 9 families. LOD scores for some serological markers (such as Gm) were uninformative, but linkage with other markers, notably the HLA antigens -A, B, -DR, -DQ, and BF on chromosome 6, could be excluded. Further studies using a comprehensive panel of gene probes to analyze DNA from families with autoimmune thyroid disease should permit the localization of the gene cluster responsible for regulating the ability to produce autoantibodies to TPO and Tg in man.

Antibodies to human thyroid peroxidase in autoimmune thyroid disease: studies with a cloned recombinant complementary deoxyribonucleic acid epitope.

Previous studies carried out by screening a lambda gt11 human thyroid cDNA library with serum samples from selected patients with Hashimoto's thyroiditis and a polyclonal antibody to porcine thyroid peroxidase (TPO) confirmed, at the molecular level, that TPO is a major component of the thyroid microsomal antigen (M). That investigation led to the isolation of a clone (C2) which encodes an 85-amino acid segment of TPO and harbors a major epitope recognized by serum from several patients with autoimmune thyroid disease that contained anti-M autoantibodies (MAb). In this study, C2 antigen that was produced as a beta-galactosidase fusion protein was used to establish an enzyme-linked immunoabsorbent assay for the detection of anti-C2 autoantibodies (C2Ab). C2Ab then were assayed in 191 patients with different autoimmune and nonautoimmune thyroid disorders, and 50 patients with nonthyroidal autoimmune diseases. The results were compared with the titers of anti-TPO antibodies (TPOAb; as detected by monoclonal antibody-assisted RIA) and MAb (as detected by passive hemagglutination). Positive C2Ab was found in the serum of 85 of 136 (63%) patients whose serum contained TPOAb and/or MAb. A significant positive correlation was found between the levels of C2Ab and those of TPOAb (r = 0.76; P less than 0.001) or MAb (r = 0.69; P less than 0.001), which was independent of the type of underlying autoimmune thyroid disorder. Low levels of C2Ab also were found in 10 of 105 (9%) serum samples that did not contain TPOAb. Western blot analysis carried out on the latter samples showed that in 2 samples the apparent C2Ab reactivity was due to the presence of antibodies reacting with beta-galactosidase. In conclusion, we confirmed the validity of screening lambda gt11 cDNA human thyroid libraries to better characterize thyroid autoantigens and demonstrated the feasibility of using recombinant proteins to establish diagnostic assays for autoantibodies.

Thyroid peroxidase and thyroid microsomal autoantibodies.

The antithyroid microsomal antibodies found in the serum of patients with autoimmune thyroid disease are directed largely, if not entirely, against thyroid peroxidase (TPO). In this study we used a highly purified, well characterized, large tryptic fragment of porcine TPO (hereafter referred to as purified porcine TPO) to examine possible differences among microsomal antibodies in patients with autoimmune thyroid disease. Antibodies against this TPO preparation and also against a synthetic peptide corresponding to residues 780-793 of the deduced sequence of the native enzyme were compared with microsomal antibodies from patients in immunoblot experiments. The antiporcine TPO and antisynthetic peptide antibodies reacted with crude preparations of human TPO. Binding of serum microsomal antibodies to purified porcine TPO was also found. Purified porcine TPO shows two fragments after gel electrophoresis under reducing conditions: a 59K fragment corresponding to the amino end of the molecule, and two approximately 30K fragments corresponding to the carboxyl end. Using an immunoblot procedure with purified porcine TPO as the antigen, we found that at least two epitopes were involved in microsomal antibody production: one associated with the 59K fragment and the other with the approximately 30K fragment(s). The distribution of serum antibodies against these epitopes differed among the patients, indicating that these antibodies comprise a heterogeneous group. Serum from patients with autoimmune thyroid disease significantly inhibited human TPO activity, raising the possibility that microsomal antibodies may contribute to the impaired thyroid function that occurs in some patients with autoimmune thyroid disease.

3beta-hydroxysteroid dehydrogenase autoantibodies are rare in premature ovarian failure.

Premature ovarian failure (POF) is a disorder of heterogeneous etiology, and autoimmunity has been suspected as one cause of POF. The steroidogenic enzyme, 3beta-hydroxysteroid dehydrogenase (3betaHSD), has been characterized as a potential autoantigen in POF as well as in insulin-dependent diabetes mellitus (type 1 diabetes). Here we studied the presence of steroid cell antibodies (SCA), autoantibodies to 3betaHSD and to two other known autoantigens in ovarian failure, steroidogenic enzymes 17alpha-hydroxylase (P450c17), and side-chain cleavage enzyme (P450scc) in POF patients and patient groups with autoimmune polyendocrinopathy syndromes type 1 and 2 (APS1 and -2), isolated Addison's disease, type 1 diabetes, and healthy controls. The SCA were found in 2 of 48 POF, 11 of 15 APS1, and 1 of 9 APS2, and autoantibodies to in vitro translated 3betaHSD protein were detected in 1 POF serum associated with Addison's disease and 3 APS1 sera. All 3betaHSD precipitating sera were also positive for SCA. However, no SCA or 3betaHSD autoantibodies were found in 38 Addison's disease, 28 type 1 diabetes, and 71 healthy control sera. In analysis of autoantibodies to P450c17 and P450scc, antibodies to these enzymes were not found in POF sera, but were found in 10 and 12 APS1 patient sera, respectively, and 1 APS2 patient serum contained anti-P450c17 antibodies. Our results show that autoantibodies to 3betaHSD in POF patients are rare and are also found in patients with APS1.

Determination at the molecular level of a B-cell epitope on thyroid peroxidase likely to be associated with autoimmune thyroid disease.

In a panel of 13 mouse monoclonal antibodies generated against native (nondenatured) human thyroid peroxidase (TPO), only 1 (monoclonal antibody 47) recognized TPO protein fragments expressed in a human TPO cDNA sublibrary. Determination of the nucleotide sequences of 18 clones recognized by monoclonal antibody 47 localized its epitope to 9 amino acids (residues 713-721) in the human TPO protein. On Western blot analysis, only TPO monoclonal antibody 47 recognized the 933-amino acid TPO molecule after denaturation and reduction of the latter, supporting the concept that the major part of the epitope is represented by a continuous portion of the TPO sequence. The binding of TPO monoclonal antibody 47 to native TPO is inhibited by immunoglobulin G in the serum of patients with autoimmune thyroid disease. The epitope for monoclonal antibody 47 defined in the present study is, therefore, part of or in the vicinity of an epitope for autoimmune thyroid disease-associated TPO antibodies.

Breast cancer in association with thyroid disorders.

BACKGROUND: The relationship between breast cancer and thyroid diseases is controversial. Discrepant results have been reported in the literature. The incidences of autoimmune and nonautoimmune thyroid diseases were investigated in patients with breast cancer and age-matched control individuals without breast or thyroid disease. METHODS: Clinical and ultrasound evaluation of thyroid gland, determination of serum thyroid hormone and antibody levels, and fine-needle aspiration of thyroid gland were performed in 150 breast cancer patients and 100 control individuals. RESULTS: The mean values for anti-thyroid peroxidase antibodies were significantly higher in breast cancer patients than in control individuals (P = 0.030). The incidences of autoimmune and nonautoimmune thyroid diseases were higher in breast cancer patients than in control individuals (38% versus 17%, P = 0.001; 26% versus 9%, P = 0.001, respectively). CONCLUSION: Our results indicate an increased prevalence of autoimmune and nonautoimmune thyroid diseases in breast cancer patients.

Proteomic analysis of human brain identifies alpha-enolase as a novel autoantigen in Hashimoto's encephalopathy.

Hashimoto's encephalopathy (HE) is a rare autoimmune disease associated with Hashimoto's thyroiditis (HT). To identify the HE-related autoantigens, we developed a human brain proteome map using two-dimensional electrophoresis and applied it to the immuno-screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI-TOF-MASS analysis, immuno-positive spots of 48 kDa (pI 7.3-7.8) detected from HE patient sera were identified as a novel autoimmuno-antigen, alpha-enolase, harboring several modifications. Specific high reactivities against human alpha-enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut-off level. Although a few HT patients showed faint reactions to alpha-enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti-alpha-enolase antibody is useful for defining HE-related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies.

Structure-activity analysis of microsomal antigen/thyroid peroxidase.

The interaction between thyroid microsomal autoantibodies and thyroid microsomal antigen/thyroid peroxidase (TPO) has been studied using both intact antigen preparations and their water-soluble trypsin fragments. In an analysis of sera from 30 patients with Graves' or Hashimoto's diseases, microsomal antibodies showed similar reactivity towards trypsin fragments (with TPO activity) and intact detergent (sodium deoxycholate, DOC)-solubilized human microsomal antigen preparations (r = 0.96). This raised the possibility that both the peroxidase-active site and the major autoantigenic site(s) of microsomal antigen were present on the same trypsin fragments. Studies with porcine TPO showed that only a few sera contained microsomal antibodies which cross-reacted strongly with the porcine preparations. Further analysis was carried out by immunoprecipitation of 125I-labelled microsomal antigen followed by SDS-PAGE and autoradiography. These studies suggest that intact human microsomal antigen (a single-chain protein with Mr = 110,000) contains an intrachain loop of amino acids formed by a disulphide bridge. Trypsin treatment cleaves the antigen close to its transmembrane section and releases a water-soluble fragment (Mr = 100,000), containing the intact disulphide-linked loop of amino acids. Further trypsin action causes cleavage of the peptide bonds within the loop in some preparations. Consequently, three major water-soluble trypsin fragments (Mr = 100,000, 73,000 and 68,000) are formed all of which contain an intact disulphide bridge and have microsomal antibody binding activities. The integrity of the disulphide bridge in intact antigen/TPO preparations and their trypsin fragments is essential for autoantibody binding activity.