T cell exhaustion is associated with the risk of papillary thyroid carcinoma and can be a predictive and sensitive biomarker for diagnosis.

BACKGROUND: The incidence of papillary thyroid carcinoma (PTC) has been steadily increasing over the past decades. Hashimoto's thyroiditis (HT) is the most common autoimmune disease, and is related to the pathogenesis of PTC. Programmed death-1 (PD-1) is currently used for the treatment of PTC, but there are very few studies on the clinical value of PD-1 in the diagnosis and targeted therapy of PTC. METHODS: The expression of T, B, NK cells and PD-1 in the peripheral blood of 132 patients with PTC (PTC group), 48 patients with nodular goiter (NG group) and 63 healthy subjects (HP group) were detected by flow cytometry. The expression of plasma T3, T4, FT3, FT4, TSH, TGAb and TPO was detected by chemiluminescence immunoassay. Among 132 PTC, 49 PTC&HT and 83 PTC&noHT were included. Among 48 NG, 10 NG&HT and 38 NG&noHT were included. The expressions of programmed death- ligand1(PD-L1) in tumor tissues of PTC group and thyroid tissues of NG group, PD-1 and CD3 in tumor infiltration lymphocyte (TIL) were detected by immunohistochemistry. RESULTS: The expression of FT3, TGAb, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ in PTC and NG was significantly higher than that in the HP group. Moreover, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ expression had significant differences between the PTC group and the NG group. In addition, the expression of TGAb, TPO, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ in PTC&HT group was significantly higher than that in the PTC&noHT group. While, the expression of B cells, CD3+PD-1+, CD3+CD4+PD-1+ and CD3+CD8+PD-1+ in PTC&HT group was higher than that in NG&HT group. PD-1 showed a significant correlation with PTC lymph node metastasis. CD3+PD-1+ and CD3+CD4+PD-1+ was higher in N1 stage than in N0 stage. Immunohistochemical results showed that the expression of PD-1, CD3 and PD-L1 in PTC was significantly higher than that in NG. CONCLUSIONS: T cell exhaustion might act as a biomarker for the differential diagnosis of PTC and NG. Patients with PTC&HT have obvious T cell exhaustion and increased expression of PD-1, PD-L1.Targeting the PD-1/PD-L1 pathway could be a new approach to prevent malignant transformation from HT to PTC&HT in the future.

Comparison of a Bridge Immunoassay with Two Bioassays for Thyrotropin Receptor Antibody Detection and Differentiation.

A rapid and fully automated chemiluminescent immunoassay for the detection of thyrotropin receptor autoantibodies (TSHR-Ab) based on a bridge technology was compared with two bioassays that measure either stimulating (TSAb) or blocking (TBAb) antibodies for the detection and differentiation of TSHR-Ab. A total of 229 patients with various thyroid disorders [151 with Graves' disease (GD), 35 with Hashimoto's thyroiditis (HT), 32 with nodular goiter, and 11 with thyroid cancer] were included. The bridge immunoassay was performed according to the manufacturer's instructions (cut-off>0.55 IU/l). TSAb and TBAb were measured with reporter bioassays. Blocking activity was defined as percent inhibition of luciferase expression relative to induction with bovine TSH alone (cut-off>34% inhibition). TSAb was reported as percentage of specimen-to-reference ratio (> 140 SRR%). The 3 TSHR-Ab assays were negative in all patients with benign euthyroid nodular goiter and differentiated thyroid cancer. In contrast, in all patients with GD, irrespective of the disease duration, TSHR-Ab positivity was present in 127 of 151 (84%) and 140 (93%) for the bridge assay and TSAb bioassay, respectively (p<0.001). Fifteen of 151 (10%) GD samples were positive in the TSAb bioassay but negative in the bridge assay. The bridge assay and the TSAb bioassay correlated positively (r=0.39, p<0.0001) in patients with GD. Both assays detected TSHR-Ab in all ten untreated hyperthyroid patients with GD. In GD patients with a duration of less than six months, 27/29 (93%) and 28 (97%) were TSHR-Ab positive with the bridge and TSAb bioassay, respectively. In comparison, TSHR-Ab were present in two of 35 (6%) and five (14%) HT patients with the bridge and TSAb bio-assay, respectively. TSHR blocking antibodies were present in one (3%) patient with HT and in two (1%) patients with GD; these two GD patients were also bridge assay positive but TSAb bioassay negative. In conclusion, the bridge immunoassay and both bioassays are highly sensitive for the detection of TSHR-Ab. The bridge assay is, however, also positive in the presence of TSHR blocking antibodies detected in a TBAb bioassay.

Trends in white blood cell and platelet indices in a comparison of patients with papillary thyroid carcinoma and multinodular goiter do not permit differentiation between the conditions.

AIM: Carcinogenesis has been related to systematic inflammatory response. Our aim was to study white blood cell and platelet indices as markers of this inflammatory response in thyroid cancer and to associate them with various clinicopathological parameters. METHODS: We included 228 patients who underwent thyroidectomy within a period of 54 months, 89 with papillary thyroid carcinoma and 139 with multinodular hyperplasia. We examined potential links between white blood cell and platelet indices on the one hand and the type thyroid pathology and various clinicopathological parameters on the other. RESULTS: No significant differences were detected between thyroid cancer and multinodular hyperplasia and no significant associations were detected with regard to lymphovascular invasion and tumor size. However, the mean platelet volume was higher in multifocal tumors, while the platelet count, plateletcrit, and platelet-to-lymphocyte ratio were increased in cases with extrathyroidal extension and in T3 tumors. Additionally, T3 tumors had lower platelet distribution width. These associations demonstrated low accuracy in predicting these pathological features, but they were found to provide a satisfying negative predictive value, with the exception of the mean platelet volume. CONCLUSIONS: White blood cell and platelet indices cannot assist in distinguishing benign goiter from thyroid cancer. However, they can provide information about tumor multifocality, extrathyroidal extension, and presence of a T3 tumor, and they may be used as a means to exclude these pathological characteristics, especially the last two, in papillary thyroid carcinoma.

B Cell Activating Factor (BAFF) and BAFF Receptor Expression in Autoimmune and Nonautoimmune Thyroid Diseases.

BACKGROUND: The B cell activating factor (BAFF) is a member of the tumor necrosis factor family, which controls the survival/proliferation of B cells and is involved in the pathogenesis of a number of autoimmune diseases. The objective of the present study was to investigate the expression of BAFF and BAFF receptor (BAFF-R) in the thyroid tissue of patients affected with autoimmune thyroid disorders (AITD) or multinodular goiter (MNG) compared with those with normal thyroids. METHODS: Immunohistochemistry was performed using a panel of antibodies against BAFF, BAFF-R, CD3, CD4, CD8, CD20, CD34, CD79a, CD1a, CD68, and CD163 on the thyroid sections of 27 patients affected with Graves' disease (GD), 23 with Hashimoto's thyroiditis (HT), 16 with nontoxic nodular goiter (NTG), and 15 with toxic nodular goiter (TG), submitted to total thyroidectomy between 2000 and 2011. RESULTS: The overall BAFF-R expression in thyrocytes was weak and not different in AITD and MNG. Conversely, a stronger BAFF expression was observed in MNG compared with AITD. BAFF and BAFF-R expression in the infiltrating lymphocytes was higher in AITD compared with MNG. Interestingly, in lymphocytes of follicular-like structures observed in HT, BAFF and BAFF-R were localized in the germinal center or in the mantle, respectively. CONCLUSIONS: This study shows that BAFF and BAFF-R are expressed in the thyrocytes derived from patients with either AITD or MNG, in addition to the expected expression of BAFF and its receptor in the infiltrating immune cells of GD and HT. These findings suggest a possible involvement of BAFF and its receptors in the pathophysiology of AITD.

Increased expression of pro-angiogenic factors and vascularization in thyroid hyperfunctioning adenomas with and without TSH receptor activating mutations.

Autonomously functioning thyroid nodules (AFTN) are known to receive an increased blood influx necessary to sustain their high rate of growth and hormone production. Here, we investigated the expression of hematic and lymphatic vases in a series of 20 AFTN compared with the contralateral non-tumor tissues of the same patients, and the transcript levels of proteins involved in the control of vascular proliferation, including the vascular endothelial growth factor (VEGF) and platelet-derived growth factors (PDGF) and their receptors and the endothelial nitric oxide synthase (eNOS). In parallel, the expression of the differentiation markers sodium/iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (Tg), and TSH receptor (TSHR) was also investigated. The data were further analyzed comparing subgroups of tumors with or without mutations in the TSHR gene. Analysis by means of CD31 and D2-40 immunostaining showed in AFTN an increased number of hematic, but not lymphatic, vessels in parallel with an enhanced proliferation rate shown by increased Ki67 staining. Quantitative RT-PCR analysis revealed an increase of VEGF, VEGFR1 and 2, PDGF-A, PDGF-B, and eNOS expression in tumor versus normal tissues. Also, higher transcript levels of NIS, TPO, and Tg were detected. Comparison of the two subgroups of samples revealed only few differences in the expression of the genes examined. In conclusion, these data demonstrate an increased expression of angiogenesis-related factors associated with an enhanced proliferation of hematic, but not lymphatic, vessels in AFTNs. In this context, the presence of TSHR mutations may only slightly influence the expression of pro-angiogenic growth factors.

Phenotypical analysis of lymphocytes with suppressive and regulatory properties (Tregs) and NK cells in the papillary carcinoma of thyroid.

CONTEXT: The immune system seems to play a key role in preventing metastasis and recurrence of thyroid cancer. T regulatory lymphocytes (Tregs) and natural killer (NK) cells play an important role in the dysfunction of the host immune system in cancer patients. OBJECTIVE: We investigated thyroid gland infiltration by Tregs and NK cells in patients with papillary thyroid cancer (PTC) and thyroid nodular goiter (TNG). The correlation between the extent of the disease and the lymphocytic infiltration of Tregs and NK cells was examined. DESIGN, SETTING, AND PARTICIPANTS: A total of 65 patients with PTC, 25 with TNG, and 50 healthy controls were studied. Blood and tissue samples from 28 patients with PTC and 13 with TNG and blood samples from the healthy controls were analyzed for T4 (CD3(+)CD4(+)), T8 (CD3(+)CD8(+)), NK (CD3(-)CD16(+)CD56(+)), and CD4(+)CD25(+)CD127(-/low) Tregs by flow cytometry (FC). Tissue samples were also analyzed for Foxp3(+) Tregs by immunohistochemistry. RESULTS: Tregs showed greater infiltration in thyroid tissue of PTC patients compared with patients with TNG (P < 0.0009 for FC and P < 0.0001 for immunohistochemistry); FC analysis of blood samples showed no difference between the groups. Flow cytometry analysis showed significantly increased NK cells in PTC tissue compared with TNG tissue (P = 0.037), whereas blood samples showed no difference. CD4(+) and CD8(+) T cells did not differ in blood and tissue samples. Increased Tregs tissue infiltration was positively correlated with advanced disease stage (P < 0.0026), whereas NK infiltration was negatively correlated (P < 0.0041). CONCLUSION: Tregs and NK cells may be important regulators of thyroid cancer progression.

High levels of circulating chemokine (C-X-C motif) ligand 11 are associated with euthyroid or subclinically hypothyroid autoimmune thyroiditis and with chemokine (C-X-C motif) ligand 10.

No data are available for chemokine (C-X-C motif) ligand 11 (CXCL11), together with CXCL10, circulating levels in autoimmune thyroiditis (AT). We measured serum CXCL11 and CXCL10 in 158 patients with newly diagnosed AT (26% with subclinical hypothyroidism), 56 euthyroid controls, and 20 patients with nontoxic multinodular goiter, all similar in gender distribution and age. CXCL11 was significantly higher in patients with AT (113±56 pg/mL) than in controls (67±16 pg/mL) or patients with multinodular goiter (75±18 pg/mL; P<0.0001). Among patients with AT, CXCL11 was significantly higher in those with a hypoechoic ultrasonographic pattern and hypothyroidism. In a multiple linear regression (MLR) model including age, thyroid volume, hypoechogenicity, hypervascularity, thyroid-stimulating hormone (TSH), and anti-thyroid peroxidase, age (P=0.009) and TSH (P<0.008) were significantly related to serum CXCL11. In an MLR model of CXCL11 (ln[pg/mL]) versus age, TSH, CXCL10 (ln[pg/mL]), TSH (P=0.028), and CXCL10 (P=0.003) were significantly and independently related to CXCL11. We first show that circulating CXCL11, together with CXCL10, is increased in patients with thyroiditis and hypothyroidism, and is related to CXCL10 levels. These results underline the importance of a Th1 immune attack in the initiation of AT.

Circulating chemokine (CXC motif) ligand (CXCL)9 is increased in aggressive chronic autoimmune thyroiditis, in association with CXCL10.

Chemokine (CXC motif) ligand (CXCL)9 (CXCL9) has been shown to be involved in autoimmune thyroid disorders, however no data are present about CXCL9 circulating levels in chronic autoimmune thyroiditis (AT) vs controls. Serum CXCL9 (and for comparison CXCL10) has been measured in patients with AT vs normal control and nontoxic multinodular goiter, and this parameter has been related to the clinical phenotype. For this study we selected 189 consecutive patients with newly diagnosed AT, 63 euthyroid controls, 30 patients with nontoxic multinodular goiter. The three groups were similar in gender distribution and age; 26% of AT patients had subclinical hypothyroidism. Serum CXCL9 was significantly higher in AT (148±110 pg/mL) than in controls (71±34 pg/mL) or patients with multinodular goiter (87±35 pg/mL) (p<0.0001). Among AT patients, CXCL9 levels were significantly higher in patients older than 50 years, those with a hypoechoic ultrasonographic pattern or with hypothyroidism. Also CXCL10 was confirmed to be associated with AT, overall in presence of hypothyroidism. In a multiple linear regression model of CXCL9 (ln[pg/mL]) vs age, thyroid volume, TSH, AbTg, AbTPO, hypoechoic pattern, the presence of hypervascularity, and CXCL10 (ln[pg/mL]), only TSH and CXCL10 (ln[pg/mL]) were significantly related to serum CXCL9 levels. We show that circulating CXCL9 is increased in patients with aggressive thyroiditis and hypothyroidism. A strong relation between circulating CXCL9 and CXCL10 has been first shown, underlining the importance of a T helper 1 immune attack in the initiation of AT.

Immunohistochemical expression of CXCR4 in thyroid carcinomas and thyroid benign lesions.

In different tumor entities, expression of the chemokine receptor 4 (CXCR4) has been linked to tumor dissemination and poor prognosis. The aim of this study was to examine the immunohistochemical expression of CXCR4 in thyroid carcinomas and thyroid benign lesions. Using monoclonal anti-CXCR4 antibody, we performed immunohistochemical staining on tissue sections from 134 cases obtained from Ruijin Hospital affiliated with Shanghai Jiaotong University School of Medicine (Shanghai, China) between 2000 and 2007. In our study, the CXCR4 expression of the thyroid carcinoma group (including 16 papillary thyroid carcinomas, 18 follicular thyroid carcinomas, 9 poorly differentiated thyroid carcinomas, and 7 medullary thyroid carcinomas) was found to be higher than in the benign lesion group (including 19 cases of Hashimoto's thyroiditis, 15 nodular goiters, and 50 follicular adenomas) (p<0.0001). Within the carcinoma group, the more malignant thyroid carcinoma group (including 9 poorly differentiated thyroid carcinomas and 7 medullary thyroid carcinomas) showed a higher ratio of CXCR4 positivity compared to the less malignant thyroid carcinoma group (including 16 papillary thyroid carcinomas and 18 follicular thyroid carcinomas) (p<0.0001). Our study suggests that CXCR4 expression might be a frequent and cancer-specific event in thyroid carcinoma, and it might be involved in malignancy transformation during the progression of thyroid carcinoma.

The incidence of Hashimoto's disease in nodular goitre: the concordance in serological and cytological findings.

OBJECTIVE: To determine the incidence of Hashimoto's disease in nodular goitre and to ascertain the degree of the concordance between serological and cytological findings. METHODS: We retrospectively reviewed data from 188 patients who underwent a fine needle aspiration biopsy of the thyroid for uninodular or multinodular goitre with a documented serological level of antithyroid peroxidase (TPO) antibodies. AntiTPO antibodies were measured by immunochemiluminescent assay (Quest Diagnostics, Madison, NJ, USA). RESULTS: The study cohort consisted of 170 female and 18 male patients with a mean (+/- SD) age of 47.8 +/- 14.9 years. AntiTPO antibodies were positive in 74 (39.36%) of the individuals and negative in 114 (60.63%). The cytodiagnoses were as follows: 5 (2.6%) cancerous, 18 (9.5%) suspicious, 12 (6.3%) inadequate, 92 (48.9%) benign and 61 (32.4%) consistent with chronic lymphocytic thyroiditis (CLT). For further analysis, we excluded all inadequate specimens. Based on the final sample of 176 patients, the sensitivity and specificity of antiTPO antibody test to detect CLT in nodular goitre were estimated to be 76.38% and 94.23% respectively. The prevalence of CLT in nodular goitre based on cytological criteria was (35.46%) compared with (31.97%) goitre based on positive antiTPO titres only. CONCLUSION: There is a high degree of concordance between serological and cytological findings of CLT in people with nodular goitres. The high prevalence of CLT in nodular goitre justifies the use of antiTPO antibodies as part of the workup in this population.

Papillary thyroid cancer, although strongly associated with lymphocytic infiltration on histology, is only weakly predicted by serum thyroid auto-antibodies in patients with nodular thyroid diseases.

OBJECTIVE: We evaluated the association between thyroid autoimmunity and thyroid cancer in a retrospective series of unselected thyroid nodules submitted to fine-needle aspiration (FNA) cytology. DESIGN: Anti-thyroid antibodies (TAb) were measured in patients with multinodular goiter (MNG) and single/isolated thyroid nodule (S/I) submitted to FNA. Thyroid lymphocytic infiltration (LI) on histology was studied in a subgroup of patients submitted to thyroidectomy; 13,021 patients were included: on cytology 622 had papillary thyroid cancer (c- PTC) and 12,399 benign thyroid nodular diseases (c-BTN). LI was evaluated in histological samples of 688 patients: 304 with PTC (h-PTC) and 384 with BTN (h-BTN). RESULTS: TAb prevalence was not different in c-BTN and c-PTC (38.7% vs 35.6%). TAb were more frequent in c-BTN than c-PTC in females with MNG (40.1% vs 32.5%, p=0.02), and in c-PTC than in c-BTN in males with S/I (31.2% vs 20.4%, p=0.02) and, although not significantly, in females younger than 30 yr (35.1% vs 30.7%). The frequency and severity of LI was significantly higher in h-PTC than h-BTN, both in MNG (82.5% vs 45.0%, p<0.001) and S/I (85.6% vs 71.0%, p<0.001), but a higher number of patients with h-PTC had negative circulating TAb, despite the presence of moderate/severe LI. CONCLUSIONS: TAb are weakly associated to PTC in males and young females, while they are more frequent in older females with BTN. The frequency and severity of LI is significantly higher in PTC than in BTN, but in cancer patients TAb are frequently negative, despite the evidence of histological thyroiditis. These data suggest that different kinds of immune response may be involved in PTC and BTN.

An outline concerning the potential use of recombinant human thyrotropin for improving radioiodine therapy of multinodular goiter.

Radioiodine ((131)I) treatment for nontoxic and toxic multinodular goiter (MNG) is an alternative therapeutic procedure used especially for patients with contraindication for surgery. Several studies have been conducted in recent years assessing the use of recombinant human TSH (rhTSH) in increasing (131)I uptake in MNGs. This procedure also decreases the activity level of the administered (131)I, changes the distribution of (131)I in the thyroid, lowers the absorption dose, and dramatically reduces the volume of the goiter (50-75% of the baseline volume). A major disadvantage, however, is the induction of hypothyroidism in a relatively large number of patients. A transient increase in thyroid volume and tenderness was noted in the first week of treatment. Also a short period (2-4 weeks) of hyperthyroidism was observed in most patients with potential consequences particularly for the elderly. Still, there has been no evidence to date that the adverse effects outweigh the positive results of using rhTSH. The use of rhTSH in benign goiter disease has not yet been approved worldwide, but its positive activity in MNG is remarkable and promising.

Characterization of thyroglobulin epitopes in patients with autoimmune and non-autoimmune thyroid diseases using recombinant human monoclonal thyroglobulin autoantibodies.

CONTEXT: Thyroglobulin (Tg) epitopes of serum Tg autoantibodies (TgAb) have been characterized using inhibition of Tg binding by human monoclonal TgAb in autoimmune thyroid diseases (AITD) [Hashimoto's thyroiditis (HT) and Graves' disease (GD)] but not in non-AITD [nontoxic multinodular goiter (NTMG) and papillary thyroid carcinoma (PTC)]. OBJECTIVE: Our objective was to compare Tg epitopes of serum TgAb from patients with AITD, non-AITD, and PTC associated with histological thyroiditis (PTC-T) using inhibition of Tg binding by four recombinant human TgAb-Fab (epitopic regions A-D). DESIGN: Inhibition of Tg binding of 24 HT, 25 GD, 19 NTMG, 15 PTC, and 25 PTC-T TgAb-positive sera by each TgAb-Fab was evaluated in ELISA. Inhibition by the pool of the four TgAb-Fab was evaluated using labeled Tg. RESULTS: Levels of inhibition were different for TgAb-Fab regions A (P = 0.001), B (0.007), and D (0.011). Inhibition by region A TgAb-Fab was significantly higher in HT, GD, and PTC-T than in NTMG and PTC patients. Inhibition levels by region B TgAb-Fab were significantly higher in HT compared with NTMG and PTC patients and in GD compared with NTMG patients. Inhibition by D region TgAb-Fab was significantly lower in NTMG than in the other groups. Inhibition by the pool ranged from 44% (NTMG) to 72% (GD). CONCLUSIONS: The pattern of Tg recognition is similar when HT patients are compared to GD and NTMG to PTC patients and differs when AITD are compared with non-AITD patients. In PTC-T patients, it is similar to that of AITD patients.

[Analysis of intracellular proapoptotic (Bax, Bak) and antiapoptotic (Bcl-2, Bcl-XL) proteins expression in thyrocytes from young patients with immune and non-immune thyroid disorders]. / Analiza ekspresji wewnatrzkomórkowych proapoptotycznych (Bax, Bak) i antyapoptotycznych (Bcl-2, Bcl-XL) bialek w tyreocytach u mlodocianych pacjentów ze schorzeniami immunologicznymi i nieimmunologicznymi gruczolu tarczowego.

Apoptosis one of the form of programmed cell death is a physiological occurrence, requisite to the correct function of every organism. This is an active process that proceeds with a participation of the cellular metabolism embracing the activation of genes and the synthesis of proteins. The signal to apoptosis can be started practically in every cell of our organism. Disturbances of the apoptosis regulation determine the essential link of the pathogenesis of many diseases, including autoimmune thyroid disorders. The aim of this study was to estimate the expression of proapoptotic (Bax, Bak) and antiapoptotic (Bcl-2, Bcl-XL) proteins in thyroid tissues from 12 patients with Graves' disease (GD), 10 with non-toxic nodular goitre (NTNG) and 10 with toxic nodular goitre (TNG). Criteria for qualification of Graves' patients: large goitre, ophthalmopathy, TRAb > 5 U/L, positive titre of anti-TPO and anti-TG antibodies and concentration of TSH <0.45 microIU/mL more the 2-3 months from onset of the disease. Detection of apoptotic proteins in thyroid follicular cells was performed by Western Blot. These analysis was confirmed by immunohistochemistry using monoclonal antibodies in DAB chromogene visuality and marked by Mayer's haematoxylin. Identification of antiapoptotic Bcl-2 and Bcl-XL molecules in the thyroid follicular cells revealed a higher expression of both proteins in patients with Graves' disease (+++; ++, respectively) in comparison to patients with NTNG (++/+; +) and TNG (++; +). The detection of proapoptotic molecules showed higher expression of Bak (++/+) and Bax (+) in Graves' thyroid tissues while Bax was in trace amount in NTNG (0/+) and TNG (0/+). We conclude that alteration in the expression of antiapoptotic and proapoptotic proteins on surface of thyroid follicular cells may play a role in the pathogenesis of thyroid autoimmune disorders. In addition, suppression of apoptosis in Graves' disease led to predominance for proliferation of thyroid follicular cells which is responsible for goitre formation.

[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.

Increased serum CXCL10 in Graves' disease or autoimmune thyroiditis is not associated with hyper- or hypothyroidism per se, but is specifically sustained by the autoimmune, inflammatory process.

OBJECTIVE: Serum CXCL10 (an interferon-gamma-inducible chemokine) levels (sCXCL10) are increased in several autoimmune conditions, including Graves' disease (GD) and autoimmune thyroiditis (AT). Longitudinal assessment of sCXCL10 in autoimmune hypo- or hyperthyroidism has not yet been performed. DESIGN AND METHODS: We longitudinally assayed sCXCL10 in the following groups: thirty-three GD and 11 toxic nodular goiter (TNG) patients when hyperthyroid (Hyper) and when reaching euthyroidism (Eu) with methimazole therapy (MMI) sixty-six AT (33 hypothyroid (Hypo) and 33 Eu) patients, basally and after reaching EU (for Hypo) with levothyroxine (L-T4) therapy twenty-two patients with thyroid cancer (CA) under L-T4-suppressive treatment, of whom 11 were re-evaluated after L-T4 withdrawal for diagnostic WBS, and 11 after recombinant TSH (rhTSH) administration thirty-three healthy controls. RESULTS: At initial evaluation, Hyper GD and AT (Hypo significantly higher than Eu) showed significantly higher mean sCXCL10 than all other groups. MMI treatment led to a significant decrease in sCXCL10 only in GD (not in TNG), while restoration of Eu, in Hypo AT, by L-T4 was not accompanied by significant sCXCL10 change. CA showed sCXCL10 comparable to controls, and both Hypo after L-T4 withdrawal and rhTSH injection had no effect on sCXCL10. CONCLUSIONS: Treatment of Hyper leads to a significant decrease in sCXCL10 only in GD, and this probably depends upon the MMI immunomodulatory effect. L-T4 correction of Hypo is not accompanied by significant modification of sCXCL10 in AT. Increased sCXCL10 is not associated with Hyper or Hypo per se, but is specifically sustained by the autoimmune inflammatory event occurring in both GD and AT.

Increase of interferon-gamma-inducible CXC chemokine CXCL10 serum levels in patients with active Graves' disease, and modulation by methimazole therapy.

BACKGROUND: CXCL10 plays an important role in the initial phases of Graves' disease (GD) and autoimmune thyroiditis (AT); however, until now, CXCL10 serum levels (sCXCL10) in patients with GD have never been evaluated in relation to thyroid function and treatment. OBJECTIVE: To evaluate sCXCL10 in GD. DESIGN: Cross-sectional. PATIENTS: One hundred and three GD, 164 AT, 20 nontoxic multinodular goitre (NTMNG), 16 toxic nodular goitre (TNG) patients and 70 healthy controls (age- and sex-matched). MEASUREMENTS: We measured sCXCL10 in patients and controls, to relate this parameter to the clinical phenotype. RESULTS: Mean sCXCL10 in GD and AT patients were comparable (122+/-81 and 133+/-102 pg/ml) and significantly higher (P<0.01) than in controls or NTMNG patients (73+/- 32 and 76+/- 25 pg/ml, respectively). Hyperthyroid GD had significantly higher sCXCL10 than euthyroid or hypothyroid GD (145+/- 92, 107+/- 56 and 105+/- 46 pg/ml, respectively; P=0.01). GD patients with untreated hyperthyroidism had higher sCXCL10 than hyperthyroid or euthyroid GD patients under methimazole (MMI) treatment (166+/-125, 124+/- 41 and 94+/- 35 pg/ml, respectively; P=0.006). Comparable sCXCL10 levels were observed in newly diagnosed untreated hyperthyroid GD patients with respect to untreated patients with relapse of hyperthyroidism after a previous MMI course (176+/-125, 155+/- 97 pg/ml, respectively). GD had similar sCXCL10 to AT and higher than TNG patients or controls (all age- and sex-matched) (144+/- 81, 149+/- 114, 101+/- 27 and 86+/- 44 pg/ml, respectively; P=0.02). CONCLUSIONS: sCXCL10 is associated with the active phase of GD in both newly diagnosed and relapsing hyperthyroid patients. The reduction in sCXCL10 in treated patients with GD may be related to the immunomodulatory effects of MMI.

[Autoimmunization and multinodular large toxic goiter therapy using repeated doses of 131I]. / Autoimmunizacja a leczenie wola guzowatego olbrzymiego nadczynnego z uzyciem powtarzalnych dawek 13lI.

The radioiodine therapy can favour and induces of autommunological reaction in thyroid gland. The aim of the study was evaluation of antithyroid autoantibodies in patients with multinodular large toxic goiter treated with repeated doses of 131I before and after therapy. Studies were conducted in 24 women (age range: 65-84 yrs) with multinodular large toxic goiters--goiter volume assessed by USG over 100 ml. Serum TSH, fT4, antithyroid antibodies (anti-TPO, anti-Tg, TSHR-Ab) levels were estimated in all patients parallel with radioiodine uptake test (after 5 and 24 hours), 131I thyroid scintigraphy and fine needle biopsy. These studies and therapy with 22 mCi 131I were repeated every 3 months. Before therapy median of thyroid volume was approximately 195 ml and during therapy gradually decreased to 110 ml after 12 months. After 12 months we found 11% of patients with hyperthyrodism, 62% of patients with euthyroidism and 27% of patients with hypothyroidism. Before radioiodine treatment aTg and aTPO presence were detected in the most of patients, but only in 5 cases above normal value. TSHRAb were detected (normal, very low values) in 16 patients. During therapy statistically significance increase of TSHRAb levels (median: from 0,27 to 0,65 after 6 months and to 0,71 IU/l after 9 months) was observed; aTPO and aTg antibodies levels showed marked tendency to rise, but without significant differences (aTPO median: from 40 IU/ml to 48 IU/ml; aTg - median: from 27 IU/ml to 46 IU/ml). During these observations we didn't find evident correlation between the levels of antithyroid antibodies, radioiodine uptake, proved reduction of goiter volume and TSH, FT4, FT3 values.

Lack of mutations in the TSHr and Gsalpha genes in TSHr antibody negative Graves' disease.

The aim of this study was to investigate whether TSHr antibody negative Graves' disease is associated with somatic mutations in the TSHr or Gsalpha genes and whether histopathologically defined thyroid lesions, i.e., hyperfunctioning adenoma, non-functioning follicular adenomas, or nodules in toxic and non-toxic multinodular goiters are associated with such mutations. No mutations but three germ-line polymorphisms were found in patients with TSHr antibody negative Graves' disease. The three polymorphisms are expected to have no or only minor effects on the signaling properties, and is not associated with altered antigenecity imposed by such mutations. Two heterozygous somatic TSHr mutations were found in two hyperfunctioning adenomas and in two toxic multinodular goiters. The lack of TSHr and Gsalpha mutations in TSHr antibody negative Graves' disease patients indicates that such mutations are neither primary nor secondary events in this disease. The results also confirm that somatic gain-of-function TSHr mutations are present in hyperfunctioning follicular adenomas and goiters, but not in non-functioning thyroid lesions.

Immunohistochemical study of progesterone receptors in thyroid gland.

OBJECTIVE: To determine the progesterone receptor status in thyroid gland. METHODS: This study was based on immunohistochemical staining of formalin fixed paraffin embedded tissues, for progesterone receptors, in 50 previously diagnosed cases of various thyroid lesions and surrounding normal thyroid tissue. RESULTS: Out of 50 cases, 8 were nodular goiter, 9 cases of adenoma, 19 papillary carcinoma, 10 follicular carcinoma and four cases were of medullary carcinoma. Surrounding normal tissue was available in 4 non-neoplastic and 21 neoplastic lesions. Overall male patients comprised 20% (10 cases) and females 80% (40 cases). Although a wide range of lesions in both the sexes including wide age range were available, none of our cases were positively stained for progesterone receptors. CONCLUSION: In contrary to earlier reports by immunohistochemical method using monoclonal mouse anti-PR antibody clone PgR 636, on formalin-fixed paraffin embedded thyroid tissues, the progesterone receptors were not detectable in our human samples. The effect of progesterone on thyroid gland may be an indirect one.