Andrzej Gardas

Epitope recognition patterns of thyroid peroxidase autoantibodies in healthy individuals and patients with Hashimoto's thyroiditis*

Objective  Thyroid peroxidase antibodies (TPOAb) are markers of autoimmune thyroid disease (AITD), including Hashimotos thyroiditis (HT), but naturally occurring TPOAb are also detectable in healthy, euthyroid individuals. In AITD, circulating TPOAb react mainly with two immunodominant regions (IDR), IDR‐A and IDR‐B. The present study was undertaken in order to compare the epitope recognition pattern of TPOAb in HT patients and healthy subjects.

Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)‐like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO‐like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR‐B, and here report our findings on the location of IDR‐A and its relationship to IDR‐B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR‐B region on the TPO model. The combination of antisera to peptides P12 (aa 549–563), P14 (aa 599–617) and P18 (aa 210–225) inhibited the binding of the mab specific for IDR‐A (mab 2) by 75%. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81·5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR‐A was found to lie immediately adjacent to IDR‐B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR‐B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model.

Proportion of Antibodies to the A and B Immunodominant Regions of Thyroid Peroxidase in Graves and Hashimoto Disease

Autoantibodies to thyroid peroxidase (TPO) predominately recognise conformational epitopes, restricted to two immunodominant regions (IDR) -A and -B. We have estimated the proportion of IDR-A and -B autoantibodies in 75 Hashimoto and 68 Graves patients sera. There were no statically significant differences between Hashimoto and Graves patients sera in the IDR-A and -B autoantibodies level, despite great differences between individual patients sera. In 75 Hashimoto patients sera, the mean value of IDR-A was 24.26%, IDR-B—52.26%, IDR (A+B)—76.66%, and to non-A, non-B regions—23.5%. In 68 Graves disease patients, the mean value of IDR-A was 24.87, IDR-B—54.29, IDR (A+B)—79.07, non-A, non-B—20.92%, at a single autoantibodies concentration 5 IU/ml. At high autoantibodies concentration (50 IU/ml) the proportion of autoantibodies to the IDR (A+B) diminished to 58% and to the non-A, non-B TPO regions increased to 42%. The autoimmune response to TPO regions outside the IDR (A+B) epitopes is stronger then previously assumed and this response is also conformation dependent.

Monozygotic twin pairs discordant for Hashimoto's thyroiditis share a high proportion of thyroid peroxidase autoantibodies to the immunodominant region A. Further evidence for genetic transmission of epitopic "fingerprints"

Thyroid peroxidase antibodies (TPOAbs) in patients with Hashimotos thyroiditis (HT) predominantly react with two immunodominant regions (IDR-A, IDR-B). Theoretically, as shown for the level of TPOAbs, the autoantibody epitopic recognition of the IDRs could be under genetic control. To examine this, we compared the distribution of TPOAb epitopic fingerprints between healthy monozygotic (MZ) co-twins and siblings to patients with clinically overt HT with a control group of euthyroid subjects, matched for sex and age, but without autoimmune thyroid disease (AITD) among their first-degree relatives. Two ELISAs based on competition with rabbit antisera were used to determine the IDR specificities in 23 patients with HT, 6 MZ co-twins, 8 siblings to patients with HT, and 11 healthy euthyroid subjects without predisposition to AITD. The fraction of TPOAbs recognizing IDR-A was 19, 18, and 9% in HT patients, MZ-co-twins, and siblings, respectively, which was higher than the 0% found in the group of healthy subjects without predisposition to AITD (p = 0.007 vs. HT; p = 0.1078 vs. MZ co-twin and p = 0.069 vs. siblings). Moreover, the IDR-A fraction differed between healthy MZ-co-twins and ordinary siblings (18% vs. 9%, p = 0.0127). In conclusion, our data indicate that the propensity to produce autoantibodies directed against the IDR-A epitope of TPO is genetically determined. This finding may have implications with respect to inheritance of autoantibody specificities in other autoimmune diseases.

Autoantibodies of IgM and IgG Class Against Eye Muscle Antigens in Patients with Graves Ophthalmopathy

The presence of autoantibodies /aab/ reacting with microsomal membrane1 or plasma membrane /AEMA/2 eye muscle antigens have been recently reported in Graves ophthalmophathy /GO/.The significance of humoral mediated immunity in GO was further supported by studies in which correlation between presence and development of ophthalmopathy and titer of anti membrane autoantibodies were established.3,4

Humoral Immunity in Graves Ophthalmopathy

There is a growing evidence that several autoantibodies might be generated in Graves ophthalmopathy (1–7) and result in diversiform clinical picture and course of the disease.

Some Evidences that Thyrotropin and Autoantibodies Binding Sites are Located on Different Polipeptide Chains of Thyroid Plasma Membrane Proteins

It is generally assumed that autoantibodies present in the sera of Graves’ patients react with thyroid cell plasma membrane at the thyrotropin /TSH/ receptor level /1-3/. A structure for the TSH receptor has been proposed as a protein built from two different subunits linked by a disulphide bridge /3/. The relative resistance of the TSH binding sites to disulphide bridges reducing agents is well known and dithiothreitol has been used to dissociate the TSH binding peptides from thyroid plasma membranes /3/. We report the high sensitivity of autoantibodies binding sites to dithiothreitol pretreatment in solubilized thyroid plasma membrane protein in contrast to relative stability of TSH binding sites. The data presented suggest a different localization of the TSH and autoantibodies binding sites and location of the autoantibodies binding sites on two polipeptide chains linked by a disulfide bridge or bridges.