Comments to SEOM clinical guidelines for the treatment of thyroid cancer

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One-tube-PCR technique for CCL2, CCL3, CCL4 and CCL5 applied to fine needle aspiration biopsies shows different profiles in autoimmune and non-autoimmune thyroid disorders.

Autoimmune thyroid diseases are characterized by lymphocytic infiltration of the thyroid gland. Chemokines are crucial in the recruitment of lymphocytes and might play an important role in the pathogenesis of autoimmune thyroid disease. The aim of this study was to test the feasibility of analysing by one-tube reverse-transcriptase polymerase chain reaction (RT-PCR) technique CC chemokine profiles in samples obtained by fine needle aspiration biopsy (FNAB). In 27 out of 35 (77%) samples, the material was sufficient for analysis and in 16 (59%) chemokines were detected, thus demonstrating the potential of this technique. Moreover, even in this small group, a statistically significant increase of CCL3 and CCL4 was found in samples from patients with autoimmune thyroid disease as compared to those with multinodular goiter. Chemokine profile measured by improved multiamplification techniques in FNAB thyroid samples may become a useful complementary tool for the management of thyroid autoimmune disease as it constitutes a source of data for research of their pathogenesis.

Thyroid autoimmune disease: demonstration of thyroid antigen-specific B cells and recombination-activating gene expression in chemokine-containing active intrathyroidal germinal centers.

Autoimmune thyroid disease--Hashimoto thyroiditis and Graves' disease--patients produce high levels of thyroid autoantibodies and contain lymphoid tissue that resembles secondary lymphoid follicles (LFs). We compared the specificity, structure, and function of tonsil and lymph node LFs with those of the intrathyroidal LFs to assess the latter's capability to contribute to autoimmune response. Thyroglobulin and thyroperoxidase binding to LFs indicated that most intrathyroidal LFs were committed to response to thyroid self-antigens and were associated to higher levels of antibodies to thyroglobulin, thyroperoxidase, and thyroid-stimulating hormone receptor. Intrathyroidal LFs were microanatomically very similar to canonical LFs, ie, they had well-developed germinal centers with mantle, light, and dark zones and each of these zones contained B and T lymphocytes, follicular dendritic and interdigitating dendritic cells with typical phenotypes. Careful assessment of proliferation (Ki67) and apoptosis (terminal dUTP nick-end labeling) indicators and of the occurrence of secondary immunoglobulin gene rearrangements (RAG1 and RAG2) confirmed the parallelism. Unexpected high levels of RAG expression suggested that receptor revision occurs in intrathyroidal LFs and may contribute to generate high-affinity thyroid autoantibodies. Well-formed high endothelial venules and a congruent pattern of adhesion molecules and chemokine expression in intrathyroidal LFs were also detected. These data suggest that ectopic intrathyroidal LFs contain all of the elements needed to drive the autoimmune response and also that their microenvironment may favor the expansion and perpetuation of autoimmune response.

Un paciente con bocio

El bocio, definido como el aumento del tamaño del tiroides, es la afección más frecuente de la glándula, tanto en su forma difusa como uni o multinodular.Prácticamente, toda la patología tiroidea puede cursar con bocio y la forma de presentación más frecuente de las neoplasias del tiroides, tanto benignas como malignas, es como un nódulo tiroideo, generalmente único. Todo ello hace que la consulta por bocio sea muy frecuente en la práctica clínica endocrinológica y confiere una importancia especial al problema del nódulo tiroideo, que siempre plantea la duda diagnóstica en cuanto a su posible malignidad.En este artículo se revisan las causas más frecuentes tanto de bocio difuso como de enfermedad nodular tiroidea y las pautas de actuación para su diagnóstico y tratamiento que se recomiendan en la actualidad. (AU)

A one-tube polymerase chain reaction protocol demonstrates CC chemokine overexpression in Graves' disease glands.

An adaptation of mixed oligonucleotide primed amplification of complementary DNA to detect the profile of CC chemokines in biological samples is presented. By introducing normalization, two correction coefficients, performing a single amplification reaction, and five parallel hybridizations, intrasample and intersample comparisons can be reliably made. This protocol of single tube PCR CC chemokine profiling was applied to tissue samples from an autoimmune thyroid condition, Graves' disease, and from a nonautoimmune condition, multinodular goiter. Results demonstrate overexpression of CC chemokines in Graves' disease, statistically significant for macrophage inflammatory protein-1alpha and -1beta, which correlated with the aberrant human leukocyte antigen class II expression by thyrocytes, as assessed by flow cytometry. Overexpression of CC chemokines probably plays a major role in determining the characteristics of the lymphocytes migrating to the thyroid gland and influences the course of the disease. The study of chemokine profile should be more informative than the study of isolated chemokines and cytokines, and as it can be applied to fine needle aspiration biopsies, it may be useful to clinical research.

[Defects of adrenal steroidogenesis in patients with hirsutism]. / Defectos de la esteroidogénesis adrenal en pacientes con hirsutismo.

BACKGROUND: To determine the frequency and the type of adrenal steroidogenic abnormalities in hirsute women. SUBJECTS AND METHODS: ACTH test was performed during follicular phase in 127 hirsute and 40 normal (control) women. Before ACTH injection we measured in serum by RIA: 17-OH-pregnenolone (17-OH-P5), 17-OH-progesterone (17-OH-P4), androstenedione (AN), cortisol (CT), 11-deoxycortisol (DCT), dehydroepiandrosterone (DHEA) and its sulphate (DHEAS), total (TT) and free (FT) testosterone, oestradiol (E2), progesterone (PR), androstenediol glucuronide (AG), LH, FSH and prolactin. After 60 min of ACTH injection 17-OH-P5, 17-OH-P4, AN, DHEA, CT and DCT were measured. Net increment of stimulated steroids and the ratios 17-OH-P5/17-OH-P4, DHEA/AN, 17-OH-P4/CT, 17-OH-P5/CT and DCT/CT were calculated. Pelvic ultrasonographic exploration was done when irregular menses were reported. RESULTS: Up to 31% of the patients presented enzymatic defects in adrenal steroidogenesis. Diagnostic criteria for enzyme defects were established. Late-onset 21-hydroxylase deficiency was diagnosed in 6 (4.5%) patients, HLA typing of these patients demonstrated that 4 out of 6 had B14-DR1. Sixteen women (12.6%) displayed a 17-OH-P4 response and the net increment 2 SD above the normal mean concentration, which are diagnostic criteria for late-onset 21-hydroxylase deficiency carriers. We diagnosed a 3 beta-hydroxysteroid dehydrogenase defect when 17-OH-P5 and DHEA responses, their net increment and the 17-OH-P5/17-OH-P4 and 17-OH-P5/CT ratios were 2 SD above the normal mean after ACTH: 14 women were diagnosed. 11 beta-hydroxylase deficiency diagnosis was made when DCT response, its net increment and the DCT/CT ratio after ACTH were 2 SD above the normal mean: 7 women were detected. Associated biosynthetic defects were described. CONCLUSIONS: One third of our patients with hirsutism presented anomalous response to ACTH, consistent with enzymatic abnormalities in adrenal steroidogenesis.

Hyperexpression of transporter in antigen processing-1 (TAP-1) in thyroid glands affected by autoimmunity: a contributing factor to the breach of tolerance to thyroid antigens?

According to the 'aberrant HLA expression' hypothesis, endocrine autoimmunity is driven by presentation of self antigens by target cells over-expressing HLA molecules. In autoimmune thyroid diseases (AITD), thyroid follicular cells (thyrocytes) over-express HLA class I and HLA class II molecules. Since efficient presentation of endogenous peptides via class I requires transporters that translocate endogenous peptides from the cytoplasm to the endoplasmic reticulum, i.e. transporters associated with antigen processing (TAP) -1 and -2, the capability of thyrocytes to express TAP and whether TAP is hyperexpressed in AITD glands are issues relevant to the above hypothesis. Results from immunofluorescence and Northern blotting studies on primary thyrocyte cultures and on a thyroid cell line demonstrate that thyrocytes express constitutively TAP-1 at a low level, and that this expression is readily induced by interferon-gamma (IFN-gamma) and to a lesser extent by IFN-alpha. In AITD, but not in non-autoimmune glands, thyrocytes hyperexpress TAP-1, as demonstrated by both immunohistopathology and flow cytometry. The cytokine pattern does not bear, as assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), a clear relationship with TAP-1 expression. These results have broad implications and suggest that the core concept of the 'aberrant HLA expression' hypothesis of endocrine autoimmunity could be incorporated in the currently prevailing view of 'autoimmunity by breach of peripheral tolerance'.

Hyperinducibility of HLA class II expression of thyroid follicular cells from Graves' disease. A primary defect?

Thyroid follicular cells (thyrocytes) from Graves' disease (GD) patients' thyroid glands express HLA class II molecules "ectopically." This phenomenon has been attributed to induction by locally produced cytokines and may be relevant to disease pathogenesis. We have compared IFN-gamma-mediated induction of HLA class II in thyrocytes from glands affected with GD and a nonautoimmune disease (MNG), to investigate a possible differential regulation of HLA expression between these two pathologies. HLA induction has been measured in primary thyrocyte cultures and control autologous macrophages stimulated or not stimulated with IFN-gamma. Comparison of flow cytometric data using an improved algorithm demonstrated that expression of HLA class II molecules is more readily induced in thyrocytes from GD than from MNG thyroid glands. This higher inducibility was parallel to a faster and stronger induction of HLA class II message in GD thyrocytes but did not correlate with the levels of HLA class II or class I originally expressed by thyrocytes in the tissue or with the degree of lymphocytic infiltration of the gland. There was no association with a particular HLA class II allele or with the presence of IFN-gamma and IL-2 in the tissue, as assessed by reverse transcription-PCR. No differences in the induction of class II were found in macrophages from each group of patients. These results suggest that an intrinsic feature of thyrocytes from GD patients is an up-regulation of HLA class II expression and that this is a characteristic that may facilitate the triggering of autoimmunity to "hyperinducible" thyroid glands.

Characterization of neural cell adhesion molecule (NCAM) expression in thyroid follicular cells: induction by cytokines and over-expression in autoimmune glands.

NCAM (CD56) is a cell surface glycoprotein of the immunoglobulin superfamily expressed on neuroendocrine and natural killer (NK) cells which has considerable molecular heterogeneity due to differential splicing and post-translational modifications. NCAM has been detected in the thyroid follicular cells (thyrocytes) immunohistologically. We report here the molecular form, the modulation by cytokines and the levels of expression in thyroid pathology. By using a panel of MoAbs to NCAM on Western blots from thyrocyte extract we have determined that these cells express the 140- and 180-kD forms of NCAM. Exposure of primary cultures of thyrocytes to interferon-gamma (IFN-gamma), and even more, to the combination of IFN-gamma plus tumour necrosis factor-alpha (TNF-alpha) induced a clear increase in the expression of NCAM as assessed by FACS analysis. NCAM expression in thyrocytes was assessed by immunofluorescence in 59 surgical specimens of thyroid glands, and was found increased in 11/17 (64%) of Graves', in 5/25 (20%) of multinodular goitre (MNG) and in occasional adenoma glands. No correlation was found with the expression of HLA class I, class II or the degree of lymphocytic infiltration scored in adjacent sections, but it was often seen in areas infiltrated by macrophages. In conclusion, NCAM is an adhesion molecule whose expression is clearly increased in thyrocytes in autoimmune glands, probably as a consequence of exposure to cytokines locally released. Since one of the forms of NCAM expressed by thyrocytes has the capability to generate intracellular signal it may play a role in normal thyroid function. In addition, NCAM may facilitate the recognition of thyrocytes by lymphocytes, particularly by NK CD56+ lymphocytes.

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.

Occurrence of thyrocyte HLA class II expression in a wide variety of thyroid diseases: relationship with lymphocytic infiltration and thyroid autoantibodies.

The proteins of the major histocompatibility system (HLA in humans) play an essential role in the regulation of immune responses due to their involvement in the presentation of antigen to T lymphocytes. Thyroid follicular cells (thyrocytes) from patients with Graves' disease and Hashimoto's thyroiditis demonstrate increased expression of HLA class I and aberrantly or inappropriately express class II antigens, a phenomenon that may play an important role in the pathogenesis of these autoimmune diseases. To establish if these changes in the expression of HLA molecules are characteristic of thyroid autoimmune disease, the immunopathological features (including class I and class II antigen expression) of 100 thyroidectomy specimens from patients with nonautoimmune thyroid disease were studied by indirect immunofluorescence, and the results compared with the findings in specimens from 14 patients with Graves' disease and 12 subjects undergoing laryngectomies for carcinoma. Increased class I product expression was found in 61% of all tissues studied, with maximal occurrence in papillary carcinomas (100%) and Graves' disease (86%), but it was also detected in 50% of the glands containing nodular lesions and in 16% of the control glands. Inappropriate class II molecule expression was found in Graves' disease (71%), hyperplastic nodules (53%), multinodular glands (44%), papillary carcinomas (38%), and 16% of the control glands. In summary, an increase in inappropriate HLA class I and class II expression was very common in nonautoimmune thyroid glands, but it generally occurred in the context of lymphocytic infiltration and thyroid autoantibodies (i.e. focal thyroiditis). Multiple correlation analyses of these 4 phenomena indicated heterogeneity in the mechanism leading to the inappropriate expression of thyrocyte class II antigens in the different conditions studied.