TSH-R expression and cytokine profile in orbital tissue of active vs. inactive Graves' ophthalmopathy patients

OBJECTIVE: From in vitro studies using cultures of orbital fibroblasts, it has become clear that cytokines play an important role in the orbital inflammation in Graves' ophthalmopathy (GO). Orbital fibroblasts seem to be the key target cells of the autoimmune attack, and they are able to express the TSH receptor (TSH-R). In vivo data on the presence of cytokines in orbital tissues are sparse, and mostly limited to samples obtained from patients with endstage, inactive GO; the same holds true for the presence of the TSH-R. The aim of the present study was to determine whether the cytokine profile and TSH-R expression differ in the active vs. the inactive stage of GO. DESIGN AND MEASUREMENTS: Orbital fat/connective tissue was obtained from six patients with active, untreated GO undergoing emergency orbital decompression, and from 11 patients with inactive GO subjected to rehabilitative decompressive surgery. The mRNA levels of various cytokines and the TSH-R were assessed by real-time polymerase chain reaction (PCR) using the LightCycler. Data are expressed as ratios (unknown mRNA/beta-actin mRNA). RESULTS: Active GO patients had much higher TSH-R expression than inactive patients: 4/0-24 (median value/range) vs. 0/0-9, P = 0.01. TSH-R expression was related to the Clinical Activity Score (r = 0.595, P = 0.015). Patients with active GO compared to those with inactive GO had higher mRNA levels of the proinflammatory cytokines interleukin-1beta (IL-1beta) (445/153-877 vs. 0/0-455, P = 0.001), IL-6 (1583/968-18825 vs. 559/0-7181, P = 0.01), IL-8 (1422/38-7579 vs. 32/0-1081, P = 0.046) and IL-10 (145/58-318 vs. 27/0-189, P = 0.002). In active GO there also existed a trend towards a predominance of T helper 1 (Th1)-derived cytokines as evident from higher IL-2 (37/0-158 vs. 0/0-68, P = 0.043), interferon-gamma (IFN-gamma) (20/0-79 vs. 0/0-16, P = 0.12) and IL-12 (2.3/0-14.8 vs. 0/0-1.6, P = 0.10) mRNAs. IL-1 receptor agonist (IL-1RA), IL-2 receptor (IL-2R), IL-3, IL-4, IL-5, IL-13, IL-18 and tumour necrosis factor-alpha (TNF-alpha) mRNAs were similar in both groups. CONCLUSIONS: These data show that at the mRNA level, TSH-R expression is largely present only during the active stages of GO. The active phase is characterized by the presence of proinflammatory and Th1-derived cytokines, whereas other cytokines, among them Th2-derived cytokines, do not seem to be linked to a specific stage of GO.     

Evaluation of endocrine tests. A: the TRH test in patients with hyperprolactinaemia

BACKGROUND: In a previous study, we determined reference values for basal and thyrotropin-releasing hormone (TRH)-stimulated plasma concentrations of prolactin (PRL). The aim of the present study was to determine the clinical usefulness of the PRL response to TRH in the work-up of patients with hyperprolactinaemia. METHODS: We studied 92 consecutive patients referred for evaluation of hyperprolactinaemia. Patients with confirmed hyperprolactinaemia were divided into three groups: group A (pharmacological hyperprolactinaemia; n=2), group B (pathological hyperprolactinaemia; n=6) and group C (all other patients). Patients in group C underwent MRI of the pituitary and were subdivided into C1 (normal pituitary on MRI; n=6), C2 (slightly abnormal MRI; n=21), and C3 (evident microadenoma or macroadenoma on MRI; n=25 and 12, respectively). The MRI was technically insufficient in four patients. Basal PRL as determined by fluoroimmunometric assay and the PRL response to 400 microg TRH were determined in all patients. RESULTS: Hyperprolactinaemia was confirmed in 83% of the referred patients. Non-response, defined as a <2.5-fold PRL increase after TRH, occurred in one patient (50%) in group A, in 66% of patients in group B and in 99% of patients in group C. Within group C, basal PRL was not different between group C1 and C2, but higher (p=0.06) in group C3. The absolute PRL increase after TRH did not differ between the three subgroups. The relative PRL increase was smaller (p=0.03) in group C3 but overlapped considerably with groups C1 and C2. All patients except one in group C were so-called non-responders. Basal PRL and absolute PRL increases after TRH correlated with the adenoma diameter on MRI (r=0.66, p=0.0002 and r=0.49, p=0.008, respectively). CONCLUSION: In patients referred for elevated serum PRL, hyperprolactinaemia should be confirmed under standardised conditions. The absolute or relative PRL increase after 400 microg TRH does not help to differentiate between patients with prolactinoma or idiopathic hyperprolactinaemia. Therefore, the TRH stimulation test is not useful in the work-up of hyperprolactinaemia.     

The biological relevance of thyroid hormone receptors in immortalized human umbilical vein endothelial cells

The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.     

Interrelationships between age, thyroid volume, thyroid nodularity, and thyroid function in patients with sporadic nontoxic goiter

PURPOSE: To test the hypothesis that during the natural history of sporadic nontoxic goiter (SNG), a diffuse goiter precedes a multinodular goiter with gradual development of autonomous thyroid function. PATIENTS AND METHODS: A cross-sectional survey of 102 consecutive patients with SNG (seven male, 95 female) was performed. Thyroid volume was measured by ultrasonography, and plasma thyroid-stimulating hormone (TSH) by a sensitive assay (TSH immunoradiometric assay). RESULTS: Patients with a multinodular goiter were older and had a larger thyroid volume than patients with a diffuse or uninodular goiter. Plasma free thyroxine (T4) and total triiodothyronine (T3) were higher and plasma TSH was lower in patients than in normal subjects. Free T4 was higher in the subgroup of patients with a multinodular goiter and a decreased TSH response to thyrotropin-releasing hormone. Plasma TSH (y, in mU/L) was negatively related to thyroid volume (x, in mL): y = 8.2x-0.667 (r = 0.578, p less than 0.001). Thyroid volume (y, in mL) was positively related to age (x, in years): y = -21.8 + 2.0x (r = 0.455, p less than 0.001); and to duration of goiter (x, in years): y = 40.6 + 2.1x (r = 0.505, p less than 0.001). The annual increase in thyroid volume was calculated at 4.5%. CONCLUSION: The data suggest a continuous growth of SNG and provide support for the concept of increasing thyroid nodularity and autonomy of thyroid function--related to increasing thyroid volume--during the natural history of this disorder.     

The application of an immunoradiometric assay of plasma thyrotropin (TSH-IRMA) in molar pregnancy

Plasma thyrotropin was measured by immunoradiometric assay (TSH-IRMA, "Sucrosep", Boots Celltech) in three patients with molar pregnancy. Two patients were hyperthyroid (increased plasma concentrations of FT4, FT4 index, and FT3 index), one patient was euthyroid (normal free thyroid hormone concentrations). TSH-IRMA values were below the detection limit (less than 0.1 mU/L) in the two hyperthyroid patients; the euthyroid patient had a TSH-IRMA value of 0.8 mU/L, within the reference range of normal subjects (0.5-3.3 mU/L). In vitro experiments demonstrated interference of the grossly elevated hCG levels in the TSH-IRMA, resulting in lower values. The observed interference was independent of ambient TSH concentrations, but related to hCG levels. We conclude that despite interference of hCG in TSH-IRMA the clinical usefulness of TSH-IRMA as a first-line test of thyroid function appears to be maintained in molar pregnancy.     

Comparison of beclomethasone dipropionate (2 and 3 mg) and prednisolone sodium phosphate enemas (30 mg) in the treatment of ulcerative proctitis. An adrenocortical approach

Twenty-three patients with attacks of distal ulcerative colitis were treated randomly with either 2 or 3 mg of topically administered beclomethasone dipropionate (BDP) or 30 mg of prednisolone sodium phosphate (PP). The effect of the steroid enemas on adrenocortical function was assessed by ACTH tests, which were performed before and after treatment. Endoscopic, clinical and histological scores were comparable in the three treatment groups in this pilot trial. Fasting cortisol in the PP group decreased significantly from 0.47 +/- 0.12 mumol/l before to 0.22 +/- 0.14 mumol/l (P less than 0.05) after therapy; in the BDP group no significant changes were found. Urinary cortisol excretion in the PP group was not detectable after therapy; in the BDP group no changes were found. It is concluded that in the topical treatment of ulcerative colitis, BDP may be preferable to PP because it exerts a promising anti-inflammatory action without affecting adrenocortical function.     

Thyrotropin receptor autoantibodies are associated with continued thyrotropin suppression in treated euthyroid Graves' disease patients

Antithyroid treatment effectively restores euthyroidism in patients with Graves' hyperthyroidism. After a few months of treatment, patients are clinically euthyroid with normal levels of thyroid hormones, but in many patients TSH levels remain suppressed. We postulated that TSH receptor autoantibodies could directly suppress TSH secretion, independently from thyroid hormone levels, via binding to the pituitary TSH receptor. To test this hypothesis, we prospectively followed 45 patients with Graves' hyperthyroidism who were treated with antithyroid drugs. Three months after reaching euthyroidism, blood was drawn for the analysis of thyroid hormones, TSH, and TSH binding inhibitory Ig (TBII) levels. After 6.7 +/- 1.5 months since start of antithyroid treatment, 20 patients still had detectable TBII levels, and 25 had become TBII negative. The two groups had similar levels of free T(4) and T(3), but TBII-positive patients had lower TSH values than TBII-negative patients: median 0.09 (range < 0.01-4.30) mU/liter vs. 0.84 (0.01-4.20; P = 0.015). In addition, TSH levels correlated only with TBII titers (r = -0.424; P = 0.004), and not with free T(4) or T(3) values. Our findings suggest that TBII suppress TSH secretion independently of thyroid hormone levels, most likely by binding to the pituitary TSH receptor.     

Evaluation of a rapid stoolantigen test for the diagnosis of Helicobacter pylori infection in Chinese patients

OBJECTIVE: To evaluate the accuracy of a rapid assay that wasdeveloped to detect Helicobacter pylori antigen in the stool,using the principle of immunochromatography, in the Chinese population. METHODS: Eligible patients without prior treatment of H.pylori were recruited. An in‐house rapid urease test (RUT) andhistology were used as the gold standard. The results of the rapidstool antigen test were compared with the gold standard. RESULTS: Valid rapid stool antigen test results for interpretationwere obtained from 94 consecutive patients (mean age: 52.5, range:22?82 years). Sensitivity, specificity, positive predictivevalue, negative predictive value and accuracy were, respectively, 77.5%,87.0%, 81.6%, 83.9% and 83.0%.The test was easy to perform and results were available within 15 min. CONCLUSION: The rapid stool antigen test using immunochromatography accuratelydiagnoses H. pylori infection in Chinese patients.