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L Bartalena, C Marcocci, and A Pinchera

Most patients with Graves' disease have some degree of ocular involvement, but only 3-5% of them develop severe ophthalmopathy (1). The reasons why only such a minority of patients with Graves' disease have severe expression of the ophthalmopathy remain to be elucidated. One possible explanation is that non-severe ophthalmopathy and severe ophthalmopathy are two different disorders with different genetic backgrounds; alternatively, they might be part of a spectrum of different conditions ranging from absent ocular involvement to most severe ophthalmopathy. In this case, external variables (i.e. environmental factors) must contribute to the nature of the expression of the disease. How important are they? How far can our intervention on environmental factors go towards reducing the risk of progression of the ophthalmopathy? In other words, to which extent, if any, is Graves' ophthalmopathy preventable? The aim of this mini-review is to address the above issues.

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C. Marcocci, A. Pinchera, and E. F. Grollman

In eukaryotes cytoplasmic pH is an important determinant of many intracellular processes and has been implicated as a regulatory signal in several cellular events (Nuccitelli & Deamer 1981). The regulation of intracellular pH (pHi) largely depends on an electroneutral, amiloride-sensitive, Na+/H+ exchange in the plasma membrane (Nuccitelli & Deamer 1981). It has recently been suggested that a stimulation of Na+/H+ exchange, producing a rapid and persistent alkalinization is associated with mitogen action in vertebrate cells (Moolenar 1986).

FRTL-5 is a continuous strain of functioning rat thyroid cells, which require TSH for their growth (Ambesi-Impiombato et al. 1980). In the present study we describe an amiloride-sensitive Na+/H+ exchange system in these cells, and we show that it plays an important role in the regulation of pHi. The pHi has been measured using the equilibrium distribution of 14C-labelled 5,5-[2-14C]dimethyl - oxazolidine -2,4 - dione (DMO) (Waddel & Butler

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E. Macchia, P. Carayon, G. F. Fenzi, S. Lissitzky, and A. Pinchera

Abstract. The purpose of this study was to develop and validate a sensitive method for evaluating adenylate cyclase stimulation by thyroid-stimulating antibodies (TSAb), based on the measurement of thyroid membrane adenylate cyclase activity in the presence of a non-hydrolyzable GTP analogue, guanyl-5'-yl imidodiphosphate (Gpp(NH)p).

The addition of Gpp(NH)p (10−5 m) produced a 10-fold increase of the sensitivity of the system for both TSH and TSAb. Immunoglobulin G preparations from sera of 30 patients with Graves' disease were tested for the adenylate cyclase stimulation either in the presence or in the absence of Gpp(NH)p: a significant stimulation was observed in 27/30 patients when the GTP analogue was added to the system, while only 20/30 patients were positive in the absence of the nucleotide. The advantage of Gpp(NH)p addition was also evident in a large series which included 57 patients with Graves' disease, 15 with Hashimoto's thyroiditis or primary myxoedema and 22 normal subjects. In fact, 88% of patients with Graves' disease resulted positive, while no significant stimulation was elicited by Hashimoto's thyroiditis, primary myxoedema and by normal immunoglobulins.

The sensitivity achieved in our system which employs thyroid plasma membranes was similar to that obtained by other investigators with the use of thyroid slices or thyroid cells in primary culture. Furthermore, methods based on thyroid plasma membranes are supposed to have a better reproducibility, since the same tissue preparation, if appropriately stored, may be used in several different tests.

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M Marino, L Chiovato, S Lisi, A Pinchera, and RT McCluskey

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.

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S Lisi, A Pinchera, RT McCluskey, L Chiovato, and M Marino

OBJECTIVE: Binding of thyroglobulin (Tg) to heparin allows efficient Tg interaction with its endocytic receptor, megalin. Rat Tg (rTg) binds to heparin using an exposed carboxyl terminal region (RELPSRRLKRPLPVK, Arg2489-Lys2503) rich in positively charged residues which is, however, not entirely conserved in human Tg (hTg) (Arg2489-Glu2503, REPPARALKRSLWVE). Here, we investigated whether and how this difference affects binding of heparin. DESIGN: To compare binding of heparin to rTg and hTg. To investigate the role of the sequence 2489-2503 using a peptide-based approach. METHODS: Binding of biotin-labeled heparin to rTg, hTg and to Tg peptides was measured in solid phase assays. RESULTS: Heparin bound to rTg with moderately high affinity (K(d): 34.2 nmol/l, K(i): 37.6 nmol/l) and to hTg with lower affinity (K(d): 118 nmol/l, K(i): 480 nmol/l) and to a lower extent. Binding was dose-dependent and saturable, and was reduced by several specific competitors (Tg itself, unlabeled heparin, lactoferrin). Heparin bound to synthetic peptides corresponding to the rat (rTgP) and to the human (hTgP) Tg sequence 2489-2503. Heparin bound to rTgP to a greater extent and with greater affinity than to hTgP. An antibody against hTgP reduced binding of heparin to intact hTg by 30%, suggesting that in hTg this region is, in part, involved in heparin binding, but also that other regions account for most of the binding. Starting from the sequence of rTgP, we designed 6 synthetic 'mutant' peptides by replacing one amino acid residue of rTgP with the corresponding residue of the sequence of hTgP. Heparin bound to 5 of 6 mutant peptides to a lower extent and with lower affinity than to rTgP. CONCLUSIONS: In spite of a reduced binding ability of the sequence 2489-2503, hTg binds to heparin, in part, using alternative, as yet unidentified, binding sites. Substitution of both positive and neutral residues within the sequence 2489-2503 reduced heparin-binding, suggesting that not only charge, but also sequence and/or conformation, may account for the heparin-binding ability of this region of Tg.

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C. Marcocci, L. Bartalena, F. Bogazzi, M. Panicucci, and A. Pinchera

Abstract. Eye disease was associated with hyperthyroidism in 202 of 221 patients with active Graves' ophthalmopathy (91.4%) and was not accompanied by thyroid hyperfunction (euthyroid Graves' disease) in the remaining 19 (8.6%). All the latter patients had some mild thyroid abnormalities (thyroid autoantibodies, negative TRH test, negative T3 suppression test, goitre). Sex distribution analysis evidenced a higher prevalence in females with a female/male ratio of 2.1 which was, however, significantly lower (P < 0.05) than that observed in control (Graves' disease patients without overt ophthalmopathy (female/male ratio = 3.4)). Patients with euthyroid Graves' disease showed a female/male ratio of 0.7. Age distribution revealed a peak prevalence in the 5th decade of life, identical to that of Graves' disease without ophthalmopathy. A close temporal relationship between the onset of hyperthyroidism and the onset of ophthalmopathy was found, since in about 85% of the patients the first ocular manifestations occurred within ±18 months around the onset of hyperthyroidism.

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G. F. Del Prete, S. Mariotti, A. Tiri, M. Ricci, A. Pinchera, and S. Romagnani

Hashimoto's thyroiditis (HT) is an autoimmune disease due to the breakdown towards self thyroid antigens, resulting in the activation of as yet incompletely defined mechanisms that lead to thyroid infiltration by both T and B lymphocytes and thyroid cell destruction.

In the studies reported here we have compared the ability of B cells from peripheral blood (PB) and thyroid infiltrate of HT patients to express surface markers of activation and to secrete thyroid autoantibody in culture. Further, T lymphocytes present in HT infiltrates have been analyzed at clonal level for their phenotype, antigen-specificity and cytolytic potential.

Phenotype and function of thyroid infiltrating B cells

In respect of peripheral blood (PB), a 2- to 3-fold increase of cells belonging to the B-cell lineage has been found in HT infiltrates (McLachlan et al. 1985). In addition, it has been recently shown that a number of thyroid B cells expressed surface markers of

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F Cetani, E Pardi, L Cianferotti, E Vignali, A Picone, P Miccoli, A Pinchera, and C Marcocci

OBJECTIVE: To report a new mutation of the multiple endocrine neoplasia type 1 (MEN1) gene in an Italian kindred. DESIGN: The study included the female proband, aged 50 years, affected by primary hyperparathyroidism, insulinoma and prolactinoma, and ten relatives. Blood samples were obtained for biochemical and genetic analyses. Clinical screening tests included serum glucose, ionized calcium, intact parathyroid hormone, GH, insulin and prolactin. The coding sequence, including nine coding exons and 16 splice sites, was amplified by PCR and directly sequenced. RESULTS: Two additional cases of primary hyperparathyroidism were identified among the paternal family members. The sequence analysis showed a heterozygous T to C transition at codon 444 in exon 9, resulting in a leucine to proline substitution (L444P) in the patient and in the two paternal family members with primary hyperparathyroidism. The L444P amino acid change was absent in 50 normal subjects. The mutation determined the loss of a BlnI restriction site of the wild-type sequence and the creation of a new restriction EcoRII site. The patient, but not her paternal affected relatives, also had a common heterozygous polymorphism (D418D) in exon 9. CONCLUSIONS: A new MEN1 mutation (L444P) in exon 9 has been identified; this substitution caused the loss of a BlnI restriction site and the creation of a new EcoRII site.

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F Aghini-Lombardi, A Pinchera, L Antonangeli, T Rago, GF Fenzi, P Nanni, and P Vitti

It is well established that iodine supplementation is effective in correcting iodine deficiency and reducing goiter prevalence. In Italy, legislation has allowed the production of iodized salt since 1972, but its consumption is on a voluntary basis. In the present study, the efficacy of legislative measures that made compulsory the availability of iodized salt in foodstores has been evaluated. Urinary iodine excretion and thyroid size, scored according to Pan American Health Organization recommendations, were determined prior to (1981) and 10 years after (1991) the introduction of legislative measures in the whole schoolchildren population residing in a restricted area of the Tuscan Appennines. Moreover, in 1991, thyroid volume was determined by ultrasonography. In 1981, mean urinary iodine excretion was 47.1±22.4 mg/kg creatinine (0.412 μmol/l) and goiter prevalence was 60%, indicating a moderate iodine deficiency. Eighty of the families subsequently used iodized salt on a regular basis; as a result of this excellent compliance, in 1991 the mean urinary iodine excretion increased to 129.7±73 mg/kg creatinine (1.24 μmol/l) and goiter prevalence dropped to 8.1%. The results of this study underline the effectiveness of iodine prophylaxis in correcting iodine deficiency and abating endemic goiter in schoolchildren, and suggest that implementation of measures that make compulsory the availability of iodized salt in foodstores overcomes the fact that there is no law governing the exclusive production and trading of iodized salt.

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E. Martino, S. Grasso, G. Bambini, G. Pardo, P. Vitti, F. Aghini-Lombardi, and A. Pinchera

Abstract. The ontogeny of thyrotropin-releasing hormone (TRH) in pancreata of human foetuses from 15–36 weeks of gestation and of infants has been studied. TRH was detectable in the pancreas of a 15 week old foetus; a progressive increase of pancreatic TRH content was observed until the 34th week of gestation, whereas a progressive decrease was found in the late period of pregnancy and in 1 year old infants. In contrast, the pancreatic insulin content showed a progressive increase during the entire pregnancy and in the first year after birth. These data indicate that TRH and insulin have different ontogenetic patterns in the human pancreas.