The use of fluorescence lifetime technology in benign and malignant thyroid tissues.

OBJECTIVE: To explore the use of fluorescence lifetime imaging microscopy in thyroid tissues, and to investigate how different thyroid lesions affect fluorescence lifetime. METHOD: Fluorescence lifetime measurements were taken of fresh frozen thyroid surgical specimens stained with fluorescein isothiocyanate tagged anti-thyroglobulin monoclonal antibodies. RESULTS: The mean fluorescence lifetime measurements in 12 patients - 3 with multinodular goitre, 4 with follicular adenoma, 4 with papillary thyroid carcinoma and 1 with follicular carcinoma - were 3.16 ns (range, 2.66-3.52 ns), 3.75 ns (range, 2.99-4.57 ns), 2.97 ns (range, 2.57-3.21 ns) and 3.61 ns, respectively. The fluorescence lifetime of follicular adenoma patients was higher than that of papillary thyroid carcinoma patients by 26 per cent (p = 0.058). The fluorescence lifetime in the follicular carcinoma patient was similar to the follicular adenoma group, but higher than in the papillary thyroid carcinoma group by 22 per cent (p = 0.01). CONCLUSION: Fluorescence lifetime measurements varied in different thyroid pathologies, possibly because of tissue-scale structural influences.

Prognostic value of change in anti-thyroglobulin antibodies after thyroidectomy in patients with papillary thyroid carcinoma

Purpose: Anti-thyroglobulin antibodies (TgAb) can be used as a surrogate tumor marker in the follow-up of papillary thyroid carcinoma (PTC). We try to determine if the change in TgAb levels in the first post-operative year is a good predictor of persistence/recurrence risk in TgAb-positive PTC patients. Methods/patients: 105 patients with PTC who underwent thyroidectomy between 1988 and 2014 were enrolled. We calculated the percentage of change in TgAb levels with the first measurement at 1-2 months after surgery and the second one at 12-14 months. Results: TgAb negativization was observed in 29 patients (27.6%), a decrease of more than 50% was observed in 57 patients (54.3%), less than 50% in 12 patients (11.4%) and in 7 patients (6.7%) the TgAb level had increased. The percentage of persistence/recurrence was 0, 8.8, 16.7 and 71.4% in each group, respectively (p < 0.001). In the multivariate analysis, only the percentage of change in TgAb showed a significant association with the risk of persistence/recurrence, regardless of other factors such as age, size and TNM stages. Conclusions: Changes in TgAb levels in the first year after surgery can predict the risk of persistence/recurrence of TgAb-positive PTC patients. Patients who achieved negativization of TgAb presented an excellent prognosis

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Novel anti-infective potential of salvianolic acid B against human serious pathogen Neisseria meningitidis.

BACKGROUND: Epidemics of meningococcal meningitis cause significant health problems especially in Sub-Saharan Africa. Novel anti-infective candidates are needed. In modern anti-adhesion therapy initial attachment of bacteria to host cells is prevented. Our unique studies have revealed anti-adhesive candidates from natural products, namely milk and berries, against Neisseria meningitidis adhesion. In the present study against N. meningitidis adhesion, a novel binding inhibitor was found; salvianolic acid B (SA-B), a polyphenol from the radix Salviae miltiorrhizae, an important part of Chinese folk medicine. METHODS: In order to test inhibition of meningococcal pili binding and anti-adhesion activity of SA-B, bovine thyroglobulin, a reference glycoprotein for meningococcal receptor was used in a microtiter plate assay. Inhibitory activity was tested by using serial dilutions of SA-B extracts of 98 and 70% purity. Results were confirmed in a HEC-1B cell dot assay and antimicrobial activity was measured by using a microbroth dilution assay. RESULTS: Almost total (93%) inhibition of pili binding, anti-adhesion, was achieved with the 70% extract of SA-B at the concentration of 0.3 mg/mL in the bovine thyroglobulin reference model. 50% binding inhibition activity was achieved with 0.6 µg/mL of the SA-B extract. Total inhibition of the pili binding to HEC-1B cells was found at the tested concentration of 0.5 mg/mL. The 98% pure SA-B resulted in weaker inhibition. At the concentration of 0.3 mg/mL 78% inhibition was achieved in the thyroglobulin model. For 50% inhibition 2.4 µg/mL of pure SA-B was needed. The difference between the binding inhibition activities (70 and 98% pure SA-B) was statistically significant (P = 0.03). Antimicrobial activity of 70% SA-B, when investigated against N. meningitidis, was detected only in relatively high concentrations. CONCLUSIONS: Our results indicate that plant SA-B may prevent meningococcal infections by inhibiting meningococcal binding and may thus have an impact on the amount of nasopharyngeal carriers of N. meningitidis. This may prevent the spreading of meningococcal infections between humans. One could conclude that SA-B and its source dried radix S. miltiorrhizae, which is an important part of Chinese folk medicine, could be valuable candidates for further research in meningococcal disease prevention.

Evidence of a combined cytotoxic thyroglobulin and thyroperoxidase epitope-specific cellular immunity in Hashimoto's thyroiditis.

CONTEXT: Hashimoto's thyroiditis (HT) is a common autoimmune disease leading to thyroid destruction due to lymphocytic infiltration. Only rare data are available regarding the recognition of specific cellular antigens, e.g. of thyroperoxidase (TPO) and thyroglobulin (Tg). OBJECTIVE: The aim of this study was to quantify and characterize TPO- and Tg-epitope-specific CD8-positive T cells of HT patients. DESIGN: Six different human leukocyte antigen (HLA)-A2 restricted, TPO- or Tg-specific tetramers were synthesized and used for measuring CD8-positive T cells in HT patients and controls. RESULTS: The frequency of peripheral TPO- and Tg-specific CD8-positive T cells was significantly higher in HLA-A2-positive HT patients (2.8 ± 9.5%) compared with HLA-A2-negative HT patients (0.5 ± 0.7%), HLA-A2-positive nonautoimmune goiter patients (0.2 ± 0.4%), and HLA-A2-positive healthy controls (0.1 ± 0.2%). The frequency of Tg-specific T cells (3.0%) was very similar to those of TPO-specific CD8-positive T cells (2.9%). Subgroup analyses revealed a steady increase of the number of epitope-specific CD8-positive T cells from 0.6 ± 1.0% at initial diagnosis up to 9.4 ± 18.3% in patients with long-lasting disease. Analyses of the number of thyroid-infiltrating cells as well as the cytotoxic capacity revealed a similar picture for TPO- and Tg-specific T cells. CONCLUSION: We here report for the first time that both antigens, TPO and Tg, are recognized by CD8-positive T cells and are involved in the thyroid destruction process leading to clinical disease manifestation.

The value of diagnostic whole-body scanning and serum thyroglobulin in the presence of elevated serum thyrotropin during follow-up of anti-thyroglobulin antibody-positive patients with differentiated thyroid carcinoma who appeared to be free of disease after total thyroidectomy and radioactive iodine ablation.

BACKGROUND: In the presence of anti-thyroglobulin antibodies (TgAb), serum thyroglobulin (Tg) might be underestimated. Therefore, the American Thyroid Association does not recommend serum Tg after thyroid hormone withdrawal or recombinant human thyrotropin administration (stimulated Tg) and diagnostic whole-body scanning (DxWBS) in TgAb-positive patients who have serum Tg values while on thyroxine (Tg-on-T4) of <1 ng/mL. The objective of this study was to determine, in patients with differentiated thyroid cancer (DTC) who appeared to be free of disease after surgery and ablative treatment, but who had positive serum TgAb, the value of performing DxWBS and obtaining serum Tg under stimulated Tg conditions. METHODS: There were 121 women and 15 men in the study. By selection criteria, all of them had total thyroidectomy with apparent complete tumor resection, remnant ablation with (131)I (1.1-5.5 GBq), and a post-(131)I therapy WBS that were negative for ectopic (131)I uptake. On assessment 8-12 months after (131)I ablation, their clinical exam needed to be normal, their Tg-on-T4 needed to be <1 ng/mL, and the test for TgAb needed to be positive. Stimulated Tg, neck ultrasound (US), and DxWBS were obtained from all patients. Patients with stimulated Tg >1 ng/mL without disease on US and DxWBS were evaluated by other imaging methods. RESULTS: In 10 (7.3%) patients, stimulated Tg was >1 ng/mL. The DxWBS revealed metastases in two of these patients, and other imaging methods showed disease in three others. Stimulated Tg was <1 ng/mL in 126 patients. DxWBS revealed metastases in three of these patients, and US detected lymph node metastases in four with a negative DxWBS. Tg stimulation combined with DxWBS revealed evidence for disease in 13 (9.5%) patients. When excluding patients with a positive US, DxWBS revealed metastases in four patients, and stimulated Tg of >1 ng/mL led to detection of persistent disease by other imaging methods in two more patients. CONCLUSIONS: Performing stimulated Tg and DxWBS at the same time seems to be useful after initial therapy in DTC patients with TgAb who do not otherwise appear to have persistent disease, even when US is negative.

Association of autonomously functioning thyroid nodules with Hashimoto's thyroiditis: study on a large series of patients.

OBJECTIVE: Autonomously functioning thyroid nodules (AFTNs) associated with Hashimoto's thyroiditis (HT) are rarely reported. This study evaluates the magnitude of such association, elaborating the clinical and biochemical characteristics of HT and AFTN. MATERIALS AND METHODS: We reviewed the records of our patients with thyroid nodules, including serum TSH, free T4 and T3, Tg-Ab, TPO-Ab, ultrasonography, Tc-99m Sodium Pertechnetate scintigraphy (performed in overt or subclinical hyperthyroid patients). HT patients with coexisting AFTN(s) (group A) were compared with patients with AFTNs alone (group B, n=267). RESULTS: 80 patients (65 women and 15 men; F:M ratio 4.3:1; age 57±15 years) had AFTN(s) and coexisting HT. Except 9 patients who were under methimazole, all had suppressed (<0.01 mU/L) or low (<0.4 mU/L) TSH; 17/71 (24%) had increased FT4 and/or FT3. Subclinical hyperthyroidism prevailed over frank hyperthyroidism in group A (76 vs. 24%), but not in group B (56 vs. 44%) ( P=0.005). Group A patients had lower serum FT3 (∼0.6 pmol/L or 9%) and FT4 (∼0.9 pmol/L or 4%) as compared to group B. The maximum diameter of the AFTN(s) was 8% smaller in group A as compared with group B, thus matching the said difference in FT3. A positive correlation between nodule size and age was found only in group B ( P=0.015). CONCLUSION: Even if difference in the size of nodules between groups A and B does not reach statistical significance, the chronic intrathyroid lymphocytic infiltration of HT may decrease the tendency of the AFTNs to grow and diminish their degree of functioning.

Balance of proliferation and cell death between thyrocytes and myofibroblasts regulates thyroid fibrosis in granulomatous experimental autoimmune thyroiditis (G-EAT).

Severe granulomatous experimental autoimmune thyroiditis (G-EAT), which progresses to fibrosis, is induced in DBA/1 mice by adoptive transfer of mouse thyroglobulin-primed and -activated spleen cells. There is extensive destruction of thyrocytes and inflammatory cell infiltration including T cells, macrophages, neutrophils, and myofibroblasts (myofbs). Suppression of transforming growth factor-beta (TGF-beta) and deficiency of interferon-gamma (IFN-gamma) inhibit fibrosis, and inflammation eventually resolves. Thyrocyte destruction in wild-type (WT) mice was a result of apoptosis, as many deoxynucleotide triphosphate nick-end labeling + apoptotic thyrocytes were present in these thyroids. The balance of apoptosis and proliferation between thyrocytes and myofbs may be important factors determining the outcome of inflammation to fibrosis versus resolution. Apoptosis and proliferation in thyrocytes versus myofbs were evaluated by dual-staining of cell-proliferating marker (Ki-67) or in situ cell death and cytokeratin or alpha-smooth muscle actin and were analyzed by confocal microscopy. Apoptotic and antiapoptotic molecules in G-EAT thyroids were detected by immunostaining. In WT thyroids, which develop fibrosis, only a few myofbs were apoptotic, and many myofbs were Ki-67+, Fas-associated death domain protein-like interleukin-1beta-converting enzyme-like inhibitory protein (FLIP)+, and Bcl-XL+. In contrast, proliferation was predominant on thyrocytes of IFN-gamma-/- mice or anti-TGF-beta-treated WT mice. These results indicate that apoptosis of inflammatory cells and regeneration of thyrocytes in IFN-gamma-/- mice and anti-TGF-beta-treated WT mice may limit development of fibrosis, whereas excessive proliferation of myofbs and loss of thyrocytes in WT mice may contribute to fibrosis.

Detection of Aspergillus fumigatus mycotoxins: immunogen synthesis and immunoassay development.

Immunological detection of secreted low molecular weight toxins represents a potentially novel means of diagnosing infection by the fungus Aspergillus fumigatus. Two such metabolites, gliotoxin and helvolic acid, were selected and conjugated to thyroglobulin for antisera generation in rabbits. Gliotoxin was initially activated using N-[p-maleimidophenyl] isocyanate (PMPI) and subsequently conjugated to S-acetyl thioglycolic acid N-hydroxysuccinimide-activated thyroglobulin, whereas helvolic acid was activated with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in the presence of thyroglobulin prior to immunisation. To facilitate subsequent antisera evaluation, both toxins were similarly conjugated to bovine serum albumin (BSA). Matrix-Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) mass spectrometry and SDS-PAGE analysis confirmed covalent attachment of toxins to BSA in the ratios of 15 and 2.4 mol per mol BSA for gliotoxin and helvolic acid, respectively. Resultant high titer antisera were capable of detecting both BSA-conjugated toxins (inhibitory concentration (IC)(50): 4-5 microg/ml). Free toxins were also detectable by competitive immunoassay, whereby 10 microg/ml free gliotoxin (30 microM) and helvolic acid (17 microM), respectively, inhibited antibody binding to cognate toxin-BSA previously immobilised on microwells. This work confirms that sensitive and specific antisera can be raised against fungal toxins and may have an application in diagnosing fungal infection.

Phosphoinositide 3-kinase inhibits megalin-mediated transcytosis of thyroglobulin across thyroid epithelial cells at a post-sorting level.

BACKGROUND: Phosphoinositide 3-kinase (PI3-K) is implicated in various cellular processes involving signaling, including intracellular trafficking. PI3-K has been shown to play a part in both receptor- and non-receptor-mediated transcytosis across cultured kidney cells and undifferentiated thyroid cells. OBJECTIVE: To investigate the role of PI3-K in transcytosis of thyroglobulin (Tg) across differentiated cultured Fisher rat thyroid cells (FRTL-5 cells) - a process known to be mediated by megalin, a member of the low-density lipoprotein receptor family. DESIGN: We studied the effect of the microbial product wortmannin, a specific inhibitor of PI3-K, on transcytosis of Tg across FRTL-5 cells. METHODS: Transcytosis experiments were performed using FRTL-5 cells cultured as tight layers on filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper cell surface. Tg was added to the upper chamber and cells were incubated at 37 degrees C. Transcytosed Tg was measured in fluids collected from the lower chamber. To study the role of PI3-K, cells were pre-incubated with wortmannin. RESULTS: Pre-incubation of FRTL-5 cells with wortmannin did not affect Tg binding and uptake, but resulted in a considerable increase in Tg transcytosis (by 40-75%, depending on the concentration of wortmannin), suggesting that PI3-K exerts an inhibitory effect on Tg transcytosis. In experiments in which a monoclonal antibody against megalin was used to reduce Tg transcytosis, pre-incubation with wortmannin did not increase Tg transcytosis from its reduced levels, indicating that PI3-K is involved in the megalin-mediated pathway. Wortmannin did not affect the extent of release of tri-iodothyronine from exogenously added Tg by FRTL-5 cells, which was used as a measure of Tg degradation in the lysosomal pathway, indicating that the effect of PI3-K on transcytosis occurs after diversion of Tg from the lysosomal pathway. CONCLUSIONS: PI3-K exerts an inhibitory role on megalin-mediated Tg transcytosis across cultured thyroid cells. PI3-K action takes place at a post-sorting level, after Tg bypassing of the lysosomal pathway.

In vivo expression of thyroid transcription factor-1 RNA and its relation to thyroid function and follicular heterogeneity: identification of follicular thyroglobulin as a feedback suppressor of thyroid transcription factor-1 RNA levels and thyroglobulin synthesis.

We used in situ hybridization to evaluate thyroid transcription factor-1 (TTF-1) RNA expression in individual follicles and related this to thyroglobulin (Tg) synthesis in vivo, as estimated by immunohistochemical analysis. We studied the thyroids of Wistar rats treated with thyroxine (T4) or propylthiouracil (PTU), each of which modulates TSH levels, but affects follicular function and Tg accumulation in the follicular lumen very differently. We show that TTF-1 RNA levels in vivo correlate directly with an increase in the cytoplasmic accumulation of Tg within the cells of individual follicles. Because TTF-1 increases Tg gene expression, RNA levels, and protein synthesis in thyroid cell cultures and because there is no correlation with TSH-increased Tg degradation within the follicular lumen, the increased cytoplasmic accumulation of Tg in vivo is interpreted to reflect TTF-1-increased Tg synthesis. Increases in serum TSH levels in the PTU or T4 treated animals did not always correlate with increases in this measure of increased Tg synthesis; and TSH levels did not always correlate with changes in TTF-1 RNA levels that would be expected to accompany increased Tg synthesis. As one possibility, this suggested there might be a hitherto unrecognized suppressor of TTF-1 RNA levels and TSH-induced Tg synthesis in individual follicles. The immunohistochemical data suggested that this suppressor might be follicular Tg itself. Supporting this possibility, we show that physiological concentrations of highly purified 19S follicular Tg decrease TTF-1 RNA levels in rat FRTL-5 thyroid cells and inhibit the action of TSH to increase Tg synthesis. We therefore suggest that follicular Tg is a feedback autoregulator of thyroid function that can counterregulate TSH actions on thyroid function in vivo and in thyroid cells in culture. We suggest this phenomenon contributes to follicular heterogeneity in vivo.

Affinity of recombinant antibody and antibody fragment binding to human thyroglobulin: potential applications in gene therapy.

Recombinant antibodies and antibody fragments are currently being produced. They can be used in vitro for the structural study of antigen-antibody interactions for instance, but their in vivo production may have applications for gene therapy of certain cancers and severe viral diseases and in developing new animal models of autoimmune disease. We report here these two types of applications using a recombinant antihuman thyroglobulin (hTg) antibody.

[A study of biological and histological criteria of thyroid autoimmunity in diffuse goiters with hyperthyroidism]. / Etude des paramètres biologiques et histologiques d'autoimmunité thyroïdienne dans les goitres diffus avec hyperthyroïdie.

Hyperthyroidism is usually classified as follows: with diffuse or with toxic plurinodular goiter, respectively considered as autoimmune and non-autoimmune thyroid disorders. This classification seems partially inadequate as signs of thyroid immunity may be found in some plurinodular toxic goiter and alternatively may by lacking in some cases of Graves' disease. These observations led us to study the intensity of intrathyroidal autoimmune process (IAP) and the levels of TBIAb, TPO- and Thyroglobulin-antibodies in 105 cases with diffuse goiter, hyperthyroidism and elevated RAIU (92 women and 13 men, age 34 +/- 11, mean +/- SD). The intensity of intrathyroidal autoimmune process (IAP) was determined by one pathologist (HT) by semi quantitative method applicable to routine clinical use. Subtotal thyroidectomy was performed because of a large goiter (n = 29), a concomitant cold nodule (n = 20), a recurrent disease (n = 18), intolerance to antithyroid drugs (n = 5) or because patients chose surgical treatment (n = 33). All cases were rendered euthyroid at the time of surgery using antithyroid drugs or iodine. The results show a lack of IAP and undetectable levels of TBIAb, TPO- and Thyroglobulin-antibodies in 10%, 11%, 25% and 47% respectively. The intensity of IAP was not different in case of first episode or recurrence of hyperthyroidism and was not related to type or duration of medical treatment. Comparison of patients with or without IAP show higher levels of TPO- and thyroglobulin-antibodies but not of TBIAb in the former group (P < 0.005). TBIAb were higher when ophthalmopathy and/or dermopathy were present vs absent (p < 0.05) and were correlated with FT4 levels (p < 0.05). The negative predictive value of TBIAb, TPO- and thyroglobulin-antibodies to predict the lack of significant IAP was 42%, 65% and 64%. The total absence of clinical, biological and histological signs of thyroid autoimmunity was found in only one case (female aged 35 with first episode of hyperthyroidism and no family history of thyroid disease) (0.9%). These results suggest that routine available criteria of thyroid immunity (including IAP) have a low specificity. It follows that they are probably inadequate to screen cases of hereditary toxic familial hyperplasia, a rare entity of still unknown prevalence.

Ras inhibits thyroglobulin expression but not cyclic adenosine monophosphate-mediated signaling in Wistar rat thyrocytes.

We previously reported that microinjection of purified Ras protein stimulated DNA synthesis in quiescent Wistar rat thyrocytes and that TSH (TSH)-stimulated DNA synthesis was Ras-dependent. In contrast to these results, microinjection of cellular or oncogenic Ras significantly reduced TSH-stimulated thyroglobulin (Tg) expression, a marker of thyrocyte differentiation. Microinjection of a dominant inhibitory Ras mutant had no effect on TSH-stimulated Tg expression. As the Tg promoter is cAMP-responsive and Ras was previously reported to interfere with entry of catalytic (C) subunit of the cAMP-dependent protein kinase into the nucleus, experiments were performed to assess the effects of Ras on cAMP-mediated signaling. Microinjection of either cellular or oncogenic Ras had no effect on TSH-stimulated entry of C subunit into the nucleus. Consistent with these data, Ras did not reduce TSH-stimulated cAMP response element binding protein phosphorylation, or cAMP response element-regulated gene expression. These results demonstrate that Ras exerts differential effects on TSH signaling; Ras increases TSH-stimulated DNA synthesis and decreases TSH-induced Tg expression. Moreover, the mechanism through which Ras induces Tg expression lies distal to entry of C subunit into the nucleus, cAMP response element binding protein phosphorylation, and cAMP response element-regulated gene expression.

Prevalence of embryotoxic factor in sera from women with unexplained recurrent abortion.

PROBLEM: The presence of embryotoxic factors in sera from women with recurrent spontaneous abortion (RSA) has been proposed as a basis for classification of unexplained RSA. To determine the prevalence of circulating embryotoxins among women with idiopathic RSA, sera from 160 women were studied using the mouse blastocyst assay. METHODS: Two-cell embryos were collected from superovulated mated (CB6F1/J mice and cultured in media supplemented with fetal bovine serum (FBS) or 10% serum at 37 degrees C with 5% CO2 and high humidity. Each assay was run in triplicate using three mice with at least five embryos from each mouse. Results were determined by calculating the average percentage atresia for each mouse. FBS, known to support embryo proliferation, was used to control in each assay. RESULTS: The prevalence of embryotoxic factors among women experiencing RSA was 24.4% (39/160). There is no correlation found between the presence of embryotoxicity and phospholipid antibodies, lupus anticoagulant, and thyroglobulin/microsomal antibodies. CONCLUSION: The embryotoxicity assay can serve as a basis for a new approach for classification of unexplained recurrent spontaneous abortion.

Thyroid autoantibodies in serum samples with abnormal TSH levels.

Serum samples with normal and abnormal levels of thyrotropin (TSH) were tested for thyroid autoantibodies. Thyroid peroxidase (TPO) antibodies were detected by a radioimmunoassay (RIA) and by an agglutination method, and thyroglobulin (Tg) antibodies by an agglutination method. Elevated levels of TPO antibodies were detected in 47% of samples with abnormal and in 12% of samples with normal levels of TSH (p < 0.001). Sixty-one percent of the biochemically hypothyroid and 26% of the biochemically hyperthyroid samples contained these antibodies (p < 0.001). Tg antibodies were only detected together with TPO antibodies. Testing of TPO antibodies from samples with abnormal TSH levels is discussed.

Interference of specific autoantibodies in the assessment of serum thyroglobulin.

Prevalence of anti-thyroglobulin autoantibody (anti-TG Ab) above 120 IU/mL was 0.238 in 491 healthy and thyroid-disorder patients (80% female, mean overall age 50 +/- 15 years) presenting to the laboratory for contrastographic, endocrinologic, or general medical purposes. It is well known that anti-TG Ab (autoantibodies to thyroglobulin) can lead to the underestimation of serum thyroglobulin (TG). In this work, recovery (REC) of TG (thyroglobulin) added to the specimen was performed in all TG assays and low REC values were considered indicative of interference. The results show that REC = 70% is a very good cut-off value both for the anti-TG Ab prevalence found in our patients and in all prevalences below 0.6. However, we used an 80% cut-off which shows higher (+11%) sensitivity and in consequence improves the clinical decision. Only 60% of low REC values are directly attributable to high levels of anti-TG Ab. In the remaining 40%, low REC values are associated with low anti-TG Ab (< 120 IU/mL). These data indicate that anti-TG Ab concentrations are not always sufficient for evaluation of interference levels since they do not take account the actual effectiveness of anti-TG Ab. In fact, the same level of anti-TG Ab may reduce REC to different degrees (up to ten times) depending on the source serum. The practical consequence of this marked variability of autoantibody activity is that in the usual double Ab immunoassays REC must be determined for each sample to avoid unsuspected interference and negative influences on the clinical decision.(ABSTRACT TRUNCATED AT 250 WORDS)

Selection of the expressed B cell repertoire by infusion of normal immunoglobulin G in a patient with autoimmune thyroiditis.

In the present study we have analyzed the changes in the expressed antibody repertoire and in temporal fluctuations of antibody levels in serum that followed infusion of normal IgG (IVIg) in a patient with autoimmune thyroiditis. Administration of IVIg resulted in the stimulation of IgM production, in alterations of expressed antibody activity in serum that could not merely be accounted for by the passive transfer of antibody specificities contained in IVIg, in transient down-regulation of B cells clones expressing a specific disease-related idiotype and in the increase in serum in recipient's autoantibodies specifically reactive with F(ab')2 fragments of IVIg. In addition, infusion of IVIg shifted the pattern of spontaneous fluctuations of autoantibody activities in the patient's serum from a pattern indicative of disconnected events in the immune network to a pattern similar to that which is consistently observed in healthy controls. These results suggest that normal IgG may modulate autoreactivity by selecting expressed antibody repertoire through V region-dependent interactions with antibodies.

Correlation of microsomal antibodies with the intensity of the intrathyroidal autoimmune process in Graves' disease.

Graves' disease is an organ-specific autoimmune disease, and intrathyroidal lymphocytes seem to be the major source of thyroid autoantibodies. Consequently, the intensity of the intrathyroidal lymphocytic infiltration is generally believed to reflect the activity of the autoimmune process. We, therefore, investigated the correlation of microsomal (enzyme immunoassay), thyroglobulin (RIA), and TSH receptor antibodies (RRA) with the degree of intrathyroidal infiltration by immunoglobulin G-producing plasma cells, activated T-cells, antigen-presenting cells, and the total number of lymphocytes. The immunocompetent cells were identified immunohistologically with monoclonal antibodies for immunoglobulins kappa and lambda, UCHL1, and the S100 antibody, respectively, in 26 thyroid glands of patients suffering from Graves' disease. The intensity of lymphocytic infiltration was determined by the point-counting method and by counting all lymphocytes and the labeled lymphocytes in 3 x 51 visual fields or 3 slides/thyroid gland. Microsomal antibodies correlated significantly (P = 0.001) with the total number of lymphocytes (r = 0.86), kappa (r = 0.71), lambda (r = 0.71), UCHL1 (r = 0.9), and S100 (r = 0.9) positive cells. These correlations were also significant for thyroglobulin antibodies. However, TSH receptor antibodies showed no significant correlations with any of the populations of immunocompetent cells. Patients with preoperatively undetectable TSH receptor or microsomal antibodies showed a broad variation of intrathyroidal infiltration by the immunocompetent cells investigated. Microsomal antibody titers, therefore, seem to reflect the intensity of the intrathyroidal autoimmune process in Graves' disease better than TSH receptor antibodies. However, the broad baseline variation in intrathyroidal infiltration observed with nondetectable thyroid antibodies will not always allow determination of the intensity of the intrathyroidal autoimmune process from microsomal or thyroglobulin antibody titers.