Proliferative activity of oxyphilic (Hurthle) cells in reactive and neoplastic thyroid lersions

The proliferative potential of oxyphilic (Hurthle) cells (HCs) present in neoplastic and non-neoplastic thyroid lesions is uncertain. To estimate the HCs ability to proliferate and to determine whether their proliferative activity correlates with the biologic behavior of different thyroid oxyphilic lesions, we selected 31 cases of chronic lymphocytic (Hashimoto’s) thyroiditis and 28 oxyphilic (Hurthle cell) thyroid tumors, including 12 adenomas and 16 carcinomas. Seven histologically normal thyroid specimens from euthyroid patients served as control tissue. The proliferative activity of HCs was evaluated by means of a double immunostaining for Ki67 and a mitochondrial antigen (which specifically recognizes oxyphilic cells). Oxyphilic cells in thyroiditis had a low proliferative activity (PA: 0.55%), although higher than that of normal thyroid parenchyma (PA: 0.06%). Neoplastic HC lesions had a mean proliferative activity of 1.56% and 6.26% in adenomas and carcinomas, respectively. A statistically significant difference was observed between proliferative activity of non-neoplastic and neoplastic lesions (p<0.01), but not within the tumor group, between adenomas and carcinomas. In addition, HC carcinomas had a statistically significant positive correlation between proliferative activity and tumor size (p<0.01) and the presence of necrosis (p<0.001).     

Thyroglobulin polymorphisms in Tunisian patients with autoimmune thyroid diseases (AITD)

The thyroglobulin (Tg) gene was reported to be linked and/or associated to autoimmune thyroid diseases (AITD) development in European Caucasian populations. Here, we attempt to replicate this finding and to evaluate the contribution of the Tg gene in the genetic susceptibility of AITD in the Tunisian population. We examined the genomic region (11.5cM) containing the Tg gene by genotyping seven microsatellite markers and four SNPs located respectively at exon 10 (Ser715Ala), exon 12 (Met1009Val), exon 21 (Ala1483Ala) and exon 33 (Arg1980Trp) in 15 Tunisian multiplex families affected with AITD including Graves' disease (GD) and Hashimoto's thyroiditis (HT) (members: 87; patients: 27 GD and 16 HT). A case-control study was performed by genotyping the Tgms2 intragenic microsatellite marker (intron 27) and four intragenic SNPs on 108 unrelated patients affected with GD and 169 normal controls. Analysis of family data did not show linkage of the thyroglobulin gene with AITD nor did analysis of case-control data show association of Tgms2 or SNPs with GD. In contrast to the European Caucasian population, we failed to detect any contribution of Tg gene in the genetic component of Tunisian AITD.     

Prevalence of autoantibodies to thyroperoxidase in patients with various thyroid and autoimmune diseases.

An original radioimmunoassay for quantitation of circulating autoantibodies (aAb) to thyroperoxidase (TPO) proved to be well suited for large scale routine testing. The present study was aimed to assess the prevalence of aAb to TPO in patients with various thyroid and autoimmune disease and, for comparison, in women referred for reproductive disorders and indication of in vitro fertilization. Anti-TPO aAb were measured in sera from 32 healthy subjects and 262 patients thoroughly investigated for thyroid dysfunction. As determined in healthy subjects, the normal level of aAb to TPO in serum ranged from 0.30 to 3.07 mg/l (of affinity-purified) anti-TPO aAb. Anti-TPO and anti-MIC aAb levels were both normal in 115 patients and correlated well (r = 0.835, P less than 0.001) in the remaining 147 patients. Coexistence of normal level of anti-TPO aAb and abnormal level of anti-MIC aAb was found in 4 patients and ascribed to a lack of specificity or sensitivity of the test for anti-MIC aAb. Coexistence of abnormal level of anti-TPO aAb and normal level of anti-MIC aAb was found in 67 patients of whom 62 presented only slightly elevated (3.1 to 10.0 mg/l) anti-TPO aAb concentration; the 5 remaining patients, all with overt thyroid autoimmune disease, showed anti-TPO levels between 10.7 to 100.7 mg/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of intercellular adhesion molecule-1 in thyroid follicular cells in autoimmune, non-autoimmune and neoplastic diseases of the thyroid gland: discordance with HLA.

The presence of intercellular adhesion molecule-1 (ICAM-1) on epithelial cells facilitates their recognition by specific T lymphocytes. To assess the possible role of ICAM-1 in the recognition of thyroid follicular cells by T cells in thyroid autoimmune disease, we investigated the expression of ICAM-1 in thyrocytes from thyroid glands affected by Graves' disease, in glands with non-autoimmune pathology and normal glands using immunofluorescence staining on cryostat sections and on dispersed cell preparations. Sequential tissue sections from glands affected by Graves' disease (n = 15), multinodular goitre (MNG, n = 26), benign nodules (n = 11), primary carcinomas (n = 12) and control thyroid glands (n = 5) were stained for ICAM-1, HLA class I, HLA class II, CD3 and thyroid peroxidase (TPO). Weak and patchy ICAM-1 expression was found in the thyrocytes of 4/15 (27%) Graves' disease and of 1/26 (4%) multinodular goitre glands. In contrast, ICAM-1 expression was detected in the thyrocytes of 5/11 (45%) benign nodules and of 8/12 (67%) thyroid carcinomas in which it was sometimes strong. Thyrocytes in the five control glands were negative. These results correlated well with flow cytometry data from 23 of these glands which showed that ICAM-1 expression in thyrocytes from Graves' patients was, when present, 'dull', while in some malignant thyrocytes it was 'bright'. In preparations of thyrocytes from Graves' disease glands we found a striking discordance between the high levels of expression of HLA class I and HLA class II and the low expression of ICAM-1. This is surprising since in vitro the expression of these three molecules is equally induced by IFN-gamma and TNF-alpha. These results suggest that additional factors are involved in the induction of the inappropriate HLA class II expression observed in the thyrocytes of glands affected by Graves' disease.     

Autoantigens in autoimmune thyroid diseases

Human thyroid peroxidase (TPO) was found to bind with both IgG and IgM from patients with autoimmune thyroid diseases. Furthermore, binding of IgG to microsomes was inhibited by TPO. Patients' IgG that passed through an antigen column packed with TPO-coupled Sepharose 4 B beads lost their hemagglutinating activity in microsome hemagglutination test. Various strains of mice were immunized with porcine TPO emulsified in complete Freund's adjuvant. Both C 57 BL/6 and C 57 BL/10 mice showed mononuclear cell infiltration in the thyroid with very high incidences of thyroiditis. The strains of mice showing thyroiditis were very different from high responder strains that were immunized with murine thyroglobulin. Cloning of human TPO cDNA was attempted to deduce the primary structure of human TPO. Using three kinds of oligonucleotides as probes, two cDNAs, 2.8 and 2.4 kb, were selected from a cDNA library constructed from mRNA purified from Graves' disease thyroid. Sequencing of cDNAs revealed that shorter cDNA lacked 171 nucleotides at the middle portion of the longer cDNA. Primer extension analysis of mRNA indicated that the full length cDNA of human TPO consists of 3,048 nucleotides and its open reading frame codes 933 amino acids.     

Costimulatory molecules and autoimmune thyroid diseases

At least two signals for proliferation and cytokine secretion by T-cells are required. The first signal is delivered through the interaction of the T-cell receptor with major histocompatibility complex (MHC) molecules expressed on the surface of antigen-presenting cells (APC). The second or costimulatory signal is delivered by cell surface molecules expressed by APC. The interaction of B7.1/B7.2 with CD28 provide the most potent costimulatory signal for T-cell activation. CD40 antigen and its ligand (CD40L) have been shown to play a major role in regulating both humoral and cellular immune responses. In autoimmune thyroid diseases autoantigen presentation could be provided by "professional" APC, such as dendritic cells, as well as "nonprofessional" APC, such as thyroid follicular cells (TFC). In fact, these cells aberrantly express MHC class II molecules in Graves' disease (GD) and Hashimoto's thyroiditis (HT), together with large amounts of MHC class I antigens: moreover, the expression of CD40 on TFC, has been demonstrated. On the other hand B7.1 has been demonstrated in HT, but not in GD TFC. This could provide in HT a local costimulatory signal for T-cell differentiation towards a type 1 cytokine secretion pattern and also result in rescue from apoptosis of infiltrating lymphocytes. The presence of ICAM-1 on the surface of HT TFC may further strengthen contact and facilitate cross-signaling between T-cells and TFC. In contrast, the absence of B7 and ICAM-1 antigens in most GD TFC may more easily be associated with anergy and apoptosis of infiltrating T-cells, preventing the perpetuation and expansion of a "destructive" autoimmune reaction.     

Vitamin D and autoimmune thyroid diseases

The role of vitamin D as an immune modulator has been emphasized in recent years, and low levels of the hormone were observed in several autoimmune diseases including multiple sclerosis and systemic lupus erythematosus. Vitamin D mediates its effect though binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. While VDR gene polymorphism was found to associate with autoimmune thyroid diseases (AITDs), few studies examined levels of vitamin D in these patients and those that did yielded conflicting results. We therefore undertook to evaluate the levels of vitamin D in patients with AITDs compared to patients with non-AITDs and healthy controls. Serum vitamin D (25-OH) levels were measured in 50 patients with AITDs, 42 patients with non-AITDs and 98 healthy subjects, utilizing the LIAISON chemiluminescence immunoassay (DiaSorin, Saluggia, Italy). Vitamin D deficiency was designated at levels lower than 10 ng/ml. Antithyroid antibodies, thyroid functions and demographic parameters were evaluated in all patients. The prevalence of vitamin D deficiency was significantly higher in patients with AITDs compared with healthy individuals (72% versus 30.6% P<0.001), as well as in patients with Hashimoto''s thyroiditis compared to patients with non-AITDs (79% versus 52% P<0.05). Vitamin D deficiency also correlated to the presence of antithyroid antibodies (P=0.01) and abnormal thyroid function tests (P=0.059). Significantly low levels of vitamin D were documented in patients with AITDs that were related to the presence of anti thyroid antibodies and abnormal thyroid function tests, suggesting the involvement of vitamin D in the pathogenesis of AITDs and the advisability of supplementation.     

Antigenic alterations in autoimmune thyroid diseases. Observations and hypotheses

In order to further define the pathobiologic changes that occur in Hashimoto's thyroiditis (HT) and Graves' disease (GD), 47 cases of these conditions and six of nodular thyroid hyperplasia were studied. Antibodies to cytokeratin, vimentin, LN-2 (a B-lymphocyte marker), UCHL-1 (a T-lymphocyte marker), and HLA-DR, and biotinylated Helix pomatia (Roman snail) lectin, were applied to paraffin sections using the avidin-biotin-peroxidase complex method. Cytokeratin was not expressed in resting epithelium or nodular hyperplasia, but was strongly displayed by injured (HT) or diffusely hyperplastic (GD) glandular tissue. Vimentin was present throughout the cytoplasm of proliferating thyrocytes but was limited to basal portions of resting cells and those of nodular hyperplasia. HLA-DR was observed in injured and hyperplastic thyroid tissue, as was N-acetyl-alpha-D-galactosamine, the target of H pomatia lectin. UCHL-1-labeled infiltrating lymphocytes were observed in both HT and GD, in areas with minimal epithelial changes, while LN-2 was observed only in association with well-formed lymphoid follicles. These findings suggest that T cells are implicated in the mechanism of both conditions, but that inflammation is not the initiating event for HT and GD; and that a "switch" in intermediate filament synthesis accompanies HLA-DR and N-acetyl-alpha-D-galactosamine expression by thyroid epithelium. Since current theories implicate intermediate filaments as intracellular mediators, we hypothesize that certain cytokeratins may be associated with gene activity governing the expression of class II histocompatibility antigens and other membrane glycoproteins in HT and GD.     

Circulating immune complexes in various thyroid diseases.

In a study of 171 patients with various thyroid diseases, circulating immune complexes (CIC), measured by a C1q solid phase radioassay, were detected in 26% of the patients as compared to 8% of the control subjects. CIC were found in 33--55% of the patients with a well defined thyroid autoimmune disorder (Hashimoto's goitre, asymptomatic thyroiditis, spontaneous myxoedema and Graves' disease) and also in the same proportion of patients with diffuse goitre. CIC were correlated to the presence of serum antibodies to microsomal thyroid antigen but not to their titre. No relationship was observed between CIC and the age or sex of the patients and the presence of exophthalmos, or between CIC and the different thyroid function tests or serum anti-thyroglobulin antibodies. CIC were found in untreated patients as well as in those treated with prednisone, methimazole or thyroxine.     

Anti-thyroid peroxidase antibodies in thyroid disorders and non-thyroid autoimmune diseases.

A new commercial method for measurement of anti-thyroid peroxidase (anti-TPO DYNOtest, Henning, Berlin) was evaluated in normal subjects and in patients with autoimmune thyroid and non-thyroid diseases, and compared to an immune fluorescence method for measurement of anti-microsomal antibodies (MicAb), and a radioimmunological method for quantifying thyroglobulin antibodies (TgAb). The majority of normal subjects had anti-TPO levels below 52 U/ml and patients with Hashimoto's thyroiditis had levels above 200 U/ml, with a good correlation to MicAb. In other autoimmune thyroid diseases the correlation was less pronounced. In non-thyroid autoimmune diseases MicAb showed falsely positive reactions in the presence of other autoantibodies, e.g. mitochondrial antibodies. The present study indicates that the anti-TPO method should probably replace measurements of MicAb for routine clinical use, thus providing a sensitive, precise, antigen specific method with the ability to reveal quantitative fluctuations. The study also indicates that TgAb could be abolished in routine diagnosis of autoimmune thyroid diseases and be reserved for special clinical situations, research purposes as well as measurement in sera before evaluation of serum thyroglobulin levels.     

HLA-D subregion expression by thyroid epithelium in autoimmune thyroid diseases and induced in vitro.

Human thyroid epithelial cells (thyrocytes) express HLA Class II molecules in autoimmune thyroid diseases (ATD). Normal thyrocytes do not express Class II, but can be induced to do so by culture with interferon-gamma (gamma-IFN). We have examined HLA-D subregion expression in sections and monolayers of thyroid by indirect immunofluorescence using appropriate monoclonal antibodies. The results indicate that, in ATD, the incidence and intensity of Class II subregion expression by thyrocytes varies between patients, and follows the pattern DR greater than DP greater than DQ. The same hierarchy is observed in cultured normal thyrocytes treated with gamma-IFN: strong induction of Class II, and of DP and DQ in particular, requires relatively high concentrations of gamma-IFN or additional factors such as thyroid stimulating hormone. These findings suggest that HLA-D subregion expression by thyrocytes in on-going ATD is determined by the levels of disease related factors in the affected tissue.     

甲状腺自身免疫病浸润单人核细胞及甲状腺上皮细胞...

Specimens of thyroid tissue from 37 cases of autoimmune thyroiditis (AT), 13 cases of thyrotoxicosis accompanied with thyroiditis (TTOT), and 23 cases of Graves' disease (GD) were analyzed by immunohistochemistry with monoclonal antibodies. The majority of infiltrating mononuclear cells were learnt to be T cells. T+4 cells were abundant in lymphoid follicles and many T+8 cells were noticed in those areas with advanced destruction. B lymphocytes were predominantly located at the germinal centers. Most importantly, DR-positive thyroid epithelial cells were significantly increased with intense lymphocytes infiltration and severe destruction of the thyroid architecture. The percentage of T cell subpopulations, B cells, macrophages were somewhat similar. Anyhow, the increase of total number of infiltrating cells and the extent of inflammatory injury were remarkable in GD, TTOT and AT. The results support the idea that some GD cases may further develop in to autoimmune thyroiditis later.     

Dendritic cells in autoimmune diseases

Subclinical autoimmune responses can be frequently detected in healthy individuals. Sustained activation of autoreactive lymphocytes is, however, required for the development of autoimmune diseases associated with ongoing tissue destruction either in single organs or generalized with multiple manifestations. Clinical and experimental evidence suggests that prolonged presentation of self antigens by dendritic cells is crucial for the development of destructive autoimmune disease. We discuss here a simplified threshold model where the key parameters for the magnitude of the autoimmune response are the amount of previously ignored self peptides presented by dendritic cells and the duration of the antigen presentation in secondary lymphoid organs. Multiple factors influence the threshold for the conversion of an autoimmune response to overt autoimmune disease. Frequent or persistent viral infections of the target organ may favor autoimmune disease by increasing the amounts of released self antigens, generating cytokine-mediated bystander activation of self-reactive lymphocytes and/or sustaining a chronic response via neoformation of lymphoid structures in the target organ.     

Expression of CD40 and apoptosis related molecules in autoimmune thyroid diseases.

Apoptosis is responsible for the loss of thyrocytes in autoimmune thyroiditis. Recent investigations into the pathogenesis of apoptosis have revealed that the important roles of suicide molecules expression on both thyrocytes and cytotoxic T-lymphocytes. To study the mechanism of thyrocyte loss in various forms of thyroiditis, we evaluated in situ expression patterns of CD40, Fas, and Fas-L on thyrocytes and infiltrating inflammatory cells by immunohistochemical staining of thyroid samples obtained from 49 patients (Graves' disease, n=10: Hashimoto's thyroiditis, n=14; nonspecific lymphocytic thyroiditis, n=11; subacute granulomatous thyroiditis, n=11; normal, n=3). The role of cytotoxic T-lymphocytes was also evaluated by analyzing the expression of granzyme B along with their phenotypic characteristics. CD40 was not expressed on thyrocytes of normal controls while they showed a diffuse expression of Fas and a scattered focal expression of Fas-L. The plump thyrocytes proximal to the inflammatory infiltrates showed more intense expressions of these three molecules in various forms of thyroiditis and a close correlation was found between CD40 and Fas-L expression on thyrocytes. Unlike Fas, which was expressed on infiltrating lymphocytes in all groups, Fas-L was not expressed on infiltrating lymphocytes, except those in subacute granulomatous thyroiditis. Granzyme B expressing activated cytotoxic T-lymphocytes occupied a negligible proportion of CD8+ T-lymphocytes in various forms of thyroiditis, and no difference was found in terms of their proportions according to the type of thyroiditis. These results show the acquisition of CD40, Fas and Fas-L molecules on thyrocytes proximal to inflammatory cell aggregates and the negligible expression of granzyme B and Fas-L on the infiltrating lymphocytes, and suggest that Fas and Fas-L mediated apoptosis of thyrocytes (fratricide) may be more important than T cell-mediated cytotoxicity in various forms of thyroiditis.     

Amylase in thyroid tissues from normal and various thyroid diseases. Biochemical and immunohistochemical study

Amylase activity was measured in thyroid tissues of various thyroid diseases and was analysed electrophoretically. Normal thyroid tissues contained significant amounts of amylase (mean +/- SD; 2.71 +/- 1.15 IU/g of tissue), and their amylase isozyme was composed of a majority of salivary type isoamylase and other peculiar isoamylase. The statistical decrease of amylase activities in tissues of Graves' disease under hyperthyroidism, thyroid carcinoma, and most of thyroid adenomas were found (Graves' disease; 1.04 +/- 0.41, carcinoma; 1.49 +/- 1.10, adenoma (except five cases with high activity); 0.88 +/- 0.49 IU/g tissue). Five of 18 cases of adenoma showed strikingly higher amylase activity in their tissues. Electrophoretical patterns of amylase isoenzymes in these five adenoma tissue were different from those of normal thyroid tissues. The cellular localization of amylase in the normal thyroid tissues and the adenoma tissues was also demonstrated immunohistochemically.