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BACKGROUND Monocarboxylate transporter 8 (MCT8) is the first thyroid hormone transporter that has been linked to a human disease. Besides genetic alterations other factors might impair MCT8 activity. AIM This study aimed at investigating whether some common drugs having a structural similarity with TH and/or whose treatment is associated with thyroid function test abnormalities, or which behave as antagonists of TH action can inhibit MCT8-mediated T3 transport. METHODS [125I]T3 uptake and efflux were measured in COS-7 cells transiently transfected with hMCT8 before and after exposure to increasing concentrations of hydrocortisone, dexamethasone, prednisone, prednisolone, amiodarone, desethylamiodarone, dronedarone, buspirone, carbamazepine, valproic acid, and L-carnitine. The mode of inhibition was also determined. RESULTS Dexamethasone significantly inhibited T3 uptake at 10 μM; hydrocortisone reduced T3 uptake only at high concentrations, i.e. at 500 and 1000 μM; prednisone and prednisolone were devoid of inhibitory potential. Amiodarone caused a reduction of T3 uptake by MCT8 only at the highest concentrations used (44% at 50 μM and 68% at 100 μM), and this effect was weaker than that produced by desethylamiodarone and dronedarone; buspirone resulted a potent inhibitor, reducing T3 uptake at 0.1-10 μM. L-Carnitine inhibited T3 uptake only at 500 mM and 1 M. Kinetic experiments revealed a noncompetitive mode of inhibition for all compounds. All drugs inhibiting T3 uptake did not affect T3 release. CONCLUSION This study shows a novel effect of some common drugs, which is inhibition of T3 transport mediated by MCT8. Specifically, dexamethasone, buspirone, desethylamiodarone, and dronedarone behave as potent inhibitors of MCT8.

Introduction: Serum thyrotropin (TSH) receptor antibodies (TRAbs) are occasionally found in patients with amiodarone-induced thyrotoxicosis (AIT), and usually point to a diagnosis of type 1 AIT (AI...

Monocarboxylate transporter 8 (MCT8) is the first thyroid hormone transporter that has been linked to a human disease. Besides genetic alterations other factors might impair MCT8 activity.This study aimed at investigating whether some common drugs having a structural similarity with TH and/or whose treatment is associated with thyroid function test abnormalities, or which behave as antagonists of TH action can inhibit MCT8-mediated T[Dexamethasone significantly inhibited TThis study shows a novel effect of some common drugs, which is inhibition of T

Monocarboxylate transporter 8 (MCT8) is the first thyroid hormone transporter that has been linked to a human disease. Besides genetic alterations other factors might impair MCT8 activity. To investigate whether some common drugs can inhibit MCT8-mediated T3 transport, [125I]T3 uptake and efflux were measured in COS-7 cells transiently transfected with hMCT8 before and after exposure to increasing concentrations of drugs. The mode of inhibition was also determined. Dexamethasone inhibited T3 uptake at 10 μM; hydrocortisone reduced T3 uptake at 500 and 1000 μM; prednisone and prednisolone were devoid of inhibitory potential. Amiodarone caused a reduction of T3 uptake by MCT8 only at the highest concentrations used (44% at 50 μM and 68% at 100 μM), this effect was weaker than that produced by desethylamiodarone and dronedarone; buspirone resulted a potent inhibitor, reducing T3 uptake even at low concentrations. L-carnitine inhibited T3 uptake only at 500 mM and 1 M. Kinetic experiments revealed a noncompetitive mode of inhibition for all compounds. All drugs inhibiting T3 uptake did not affect T3 release. This study shows a novel effect of some common drugs, which is inhibition of T3 transport mediated by MCT8. Specifically, dexamethasone, buspirone, desethylamiodarone, dronedarone behave as potent inhibitors of MCT8.

Background Premature ovarian insufficiency (POI) is an ovarian defect characterized by primary or secondary amenorrhea, hypergonadotropism and hypoestrogenism which occurs before the age of 40 years with a major genetic component. In this study we performed clinical evaluation and genetic analysis of a group of 18 patients with POI. The study involved 18 consecutive women with POI. Karyotiping and genetic analysis for research of mutations in GDF9 (Growth Differentation Factor 9) and BMP15 (Bone morphogentic protein 15) genes and FMR1 (Fragile X Mental Retardation 1) premutation were carried out. In vitro functional study of the novel BMP15 mutation was performed using COV434 (Human ovarian granulosa tumour cells 434) cells of ovarian granulosa, which consistently express BMP responsive element, and luciferase reporter assay. Results Three patients (17%) had a family history of POI. Ten patients (56%) had a family history of autoimmune diseases and nine patients (50%) showed a personal history of one or more autoimmune diseases. Of patients for whom morphological assessment was available, almost half (44%) had poor follicle assets or small ovaries’s size at pelvic US. Two patients (13%) showed reduced bone density at DEXA (Dual Energy X-ray Absorptiometry). All the women had normal female kariotype and no mutations in the GDF-9 gene or FMR1 premutations were found. A novel heterozygous mutation c.406G > C (V136L) of BMP15 gene was identified in one patient. After transfection in COV434 cells, BMP15 variant showed a significantly reduced luciferase activity compared to wild type. Conclusions POI is a multifactorial disease with several health implications. Autoimmunity and genetics represent the most common aetiology. We identified and characterized a novel BMP15 mutation, providing an additional elucidation of molecular basis of this complex disorder.

Purpose Toxic multinodular goiter is a heterogeneous disease associated with hyperthyroidism frequently detected in areas with deficient iodine intake, and functioning and non-functioning nodules, characterized by increased proliferation but opposite functional activity, may coexist in the same gland. To understand the distinct molecular pathology of each entity present in the same gland, the gene expression profile was evaluated by using the Affymetrix technology. Methods Total RNA was extracted from nodular and healthy tissues of two patients and double-strand cDNA was synthesized. Biotinylated cRNA was obtained and, after chemical fragmentation, was hybridized on U133A and B arrays. Each array was stained and the acquired images were analyzed to obtain the expression levels of the transcripts. Both functioning and non-functioning nodules were compared versus healthy tissue of the corresponding patient. Results About 16% of genes were modulated in functioning nodules, while in non-functioning nodules only 9% of genes were modulated with respect to the healthy tissue. In functioning nodules of both patients and up-regulation of cyclin D1 and cyclin-dependent kinase inhibitor 1 was observed, suggesting the presence of a possible feedback control of proliferation. Complement components C1s, C7 and C3 were down-regulated in both types of nodules, suggesting a silencing of the innate immune response. Cellular fibronectin precursor was up-regulated in both functioning nodules suggesting a possible increase of endothelial cells. Finally, Frizzled-1 was down-regulated only in functioning nodules, suggesting a role of Wnt signaling pathway in the proliferation and differentiation of these tumors. None of the thyroid-specific gene was deregulated in microarray analysis. Conclusion In conclusion, the main finding from our data is a similar modulation for both kinds of nodules in genes possibly implicated in thyroid growth.

Background: Peach gibberellin-regulated protein (peamaclein) has recently emerged as a relevant food allergen in cypress pollen–hypersensitive patients. Objective: We investigated monosensitization to peamaclein among Italian cypress pollen–allergic patients. Patients: A total of 835 cypress pollen–hypersensitive patients from 28 Italian allergy centers underwent a thorough work-up to determine food-allergic reactions and performed skin prick testing with a commercial peach extract containing peamaclein. IgE to rPru p 3 was measured in peach reactors, and those with negative results were enrolled as potentially monosensitized to peamaclein. IgE reactivity to rPru p 7 was evaluated using immunoblot and an experimental ImmunoCAP with rPru p 7. Results: Skin prick tests were positive to peach in 163 patients (19.5%); however, 127 (77.9%) were excluded because they reacted to Pru p 3. Twenty-four patients (14.7%) corresponding to 2.8% of the entire study population) were considered potentially monosensitized to peamaclein. No geographic preference was observed. Seventeen of the 24 patients (70.8%) had a history of food allergy, mainly to peach (n=15). Additional offending foods included other Rosaceae, citrus fruits, fig, melon, tree nuts, and kiwi. On peach immunoblot, only 3 of 18 putative peamaclein–allergic patients reacted to a band at about 7 kDa; an additional 4 patients reacted at about 50-60 kDa. Ten of 18 patients (56%) had a positive result for Pru p 7 on ImmunoCAP. Conclusion: Allergy and sensitization to peamaclein seem rare in Italy. Most patients react to peach, although other Rosaceae fruits and several citrus fruits may also be offending foods. Peach and cypress pollen probably also share cross-reacting allergens other than peamaclein.

Summary The clinical usefulness of two commercial peach extracts for SPT (by Lofarma SpA and ALK-Abello, respectively) was compared in a multicenter study carried out in Italy. Peach allergic patients were tested with the two extracts in parallel and underwent the detection of IgE specific for all three peach allergens currently available (Pru p1, Pru p3, and Pru p4, respectively). The two extracts were almost identical in terms of sensitivity and specificity, being able to detect virtually all patients sensitized to stable peach allergens (lipid transfer protein (LTP) and, presumably, peamaclein) but scoring negative in patients exclusively sensitive to labile allergens (either PR-10 and/or profilin). Thus, the two extracts represent an excellent tool to carry out a preliminary component-resolved diagnosis of peach allergy at the first patient visit.

Nodular goiter in patients from areas of iodine deficiency is due to the growth of follicular and endothelial cells, involving different vascular-related growth factors in its pathogenesis. The aim of our study was to examine the association of known single polymorphisms of vascular endothelial growth factor-A [VEGF-A], VEGF receptor-2 [VEGFR-2] and hypoxia-inducible factor-1α [HIF-1α] genes or their genetic interactions with the risk of nodular goiter development. 116 normal subjects, without any thyroid disease, and 108 subjects with nodular goiter [subjects with goiter and at least one thyroid nodule of > 1 cm of maximum size and in absence of signs of autoimmunity] were selected from a homogeneous population living in a mild iodine deficiency geographic area. Analyses were performed on germline DNA obtained from blood samples and VEGF-A rs3025039, VEGFR-2 rs2071559, and HIF-1αrs11549465 SNPs were investigated by real-time PCR technique. The multifactor dimensionality reduction [MDR] methodology was applied to investigate the genetic interaction between SNPs. Hardy–Weinberg equilibrium was performed. None of the studied polymorphisms were individually associated with a higher risk to develop nodular goiter [P > 0.05]. The combination of the VEGF-A rs3025039 and VEGFR-2 rs2071559 polymorphisms had the highest accuracy of 0.58 [P = 0.018] and the interaction of some genotypes was significantly associated with the risk of nodular goiter development. Our results support a genetic interaction between the VEGF-A rs3025039 and VEGFR-2 rs2071559 polymorphisms as a predictor of the risk to develop nodular goiter in subjects coming from an area with mild iodine deficiency.

Soy food health effects are due to isoflavone (ISF) components functioning as phytoestrogens similar to natural estrogen. Epidemiological data suggest a role of estrogens in pathogenesis of thyroid diseases explaining their prevalence in women. Aim of this study was to determine in vitro effects of isoflavones, soybean extract and Cimicifuga extract on iodide uptake, a crucial step in thyroid hormone biosynthesis, by using a differentiated TSH-dependent cell line (FRTL5). Estradiol didn’t influence iodide uptake and sodium-iodide symporter (NIS) mRNA expression in FRTL-5 cells. None of analyzed glycoside or aglycone isoflavone determined a significant inhibition of iodide uptake, while both isoflavone rich and triterpene rich extracts determined a significant reduction of iodide uptake at the higher doses. While soybean and Cimicifuga extracts inhibited iodide uptake at high doses, none of analyzed isoflavone exerted this effect. These elevated doses, however, are never reached in the blood following intake of food supplements.

One of the best indicators of adrenal gland dysfunction is the level of free cortisol measured in the 24-h urine (UFC) which faithfully reflects the level of biologically active serum cortisol not subjected to circadian variations. Liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) is a sensitive, accurate and precise method recently available in routine laboratories that could remedy interference problems of immunoassays. In this study, a literature reference range for UFC measured by LC–MS–MS was verified, and UFC values measured by LC–MS–MS and immunoassay were compared. Immunometric UFC measurement was performed by ACCESS CORTISOL assay without preliminary extraction, using Beckman Coulter UniCel DxI 600 highly automated platform. Liquid chromatography–tandem mass spectrometry UFC measurement was performed by a home-made validated method using cortisol-D4 as internal standard with preliminary deproteinization of urinary samples by centrifugal filter and injection on reverse-phase column. Cortisol was analyzed in positive ion mode with an ESI interface. The reference interval from literature (11–70 μg/day) was confirmed by results obtained for healthy study group. Comparison study of the two methods highlighted a constant and proportional systematic error with a general tendency to overestimate results for the in-use method. In conclusion, the direct immunometric method overestimates UFC results with respect to liquid chromatography–tandem mass spectrometry which represents the reference method. The literature reference range 11–70 μg/day was confirmed and can be adopted by our lab that will shift all UFC tests performed in routine to the mass spectrometry-based method, satisfying clinicians’ request.

Nonautoimmune subclinical hypothyroidism (NSH) is characterized by elevated serum TSH in presence of normal thyroid hormone levels and absence of anti-thyroid antibodies. As result of a genomic-wide study, a strong association between three polymorphic variants in intron 1 of human PDE8B gene (rs4704397, rs6885099, rs2046045) and serum TSH has been reported in euthyroid subjects.The aim of this study was to evaluate frequency and effects on serum TSH of PDE8B gene polymorphisms in patients with sporadic NSH and verify if differences in serum TSH levels are associated to these polymorphic variants.A total of 58 Italian selected patients affected by NSH, with elevated serum TSH, normal FT3 and FT4 and without TSHr gene mutations, were subjected to genotyping for specific single nucleotide polymorphism of PDE8B gene.In all patients, the integrity of TSH receptor gene was attested. The ancestral allele associated with increased serum TSH was present in 42/58 patients (72.4 %) for rs4704397, in 42/58 patients (72.4 %) for rs6885099 and in 44/58 patients (75.9 %) for rs2046045. However, similar values of serum TSH were detected in patients with minor or major allele for each polymorphism.A prevalence of the minor allele of PDE8B gene polymorphism associated with elevated serum levels of TSH was demonstrated in patients affected by sporadic NSH; however, significant differences in circulating TSH in patients with minor or major alleles for each polymorphism were not identified demonstrating the lack of association between the polymorphisms and serum TSH levels in these patients.

Congenital hypothyroidism (CH) due to thyroglobulin (TG) deficit is an autosomal recessive disease (OMIM #274700) characterized by hypothyroidism, goiter, low serum TG, and a negative perchlorate discharge test. The aim of this study was to perform the genetic analysis of the TG gene in two sisters born from consanguineus parents and affected by CH and low serum TG levels. The index patient and her sister were identified at neonatal screening for CH and treated with L-thyroxine (L-T4). After discontinuation of L-T4 therapy, hypothyroidism was confirmed, serum TG was undetectable, and no organification defect after 123I scintigraphy and perchlorate test was shown; thyroid ultrasound showed a eutopic gland of normal size. DNA was extracted from peripheral white blood cells of the two sisters and the father. All 48 exons of TG gene were amplified by polymerase chain reaction and subjected to direct sequencing. A novel homozygous point mutation in exon 10 of TG gene was identified in the patient and her sister. The mutation determined a stop codon at position 768 (R768X) resulting in an early truncated protein or in the complete absence of the protein. The father (euthyroid) was heterozygous carrier of the mutation. Conclusion: Genetic analysis of TG gene was performed in two sisters affected by CH. A novel point mutation of the TG gene determining a stop codon at position 768 of the protein was identified. The early truncated nonfunctioning protein or the absence of the protein due to the premature degradation of abnormal mRNA may be responsible of the observed phenotype.

Congenital hypothyroidism (CH) associated with goiter or a gland of normal size has been linked to dual oxidase 2 (DUOX2) mutations in the presence of iodide organification defect.Thirty unrelated children with CH or subclinical hypothyroidism (SH) identified during infancy with a eutopic thyroid gland, coming from our Screening Centre for CH or referred from other regions of Italy, were studied with the perchlorate discharge test to identify organification defects. Eleven children with iodide organification defect were considered for the genetic analysis of TPO, DUOX2, and dual oxidase maturation factor 2 (DUOXA2) genes.Eight children with CH and three with SH and eutopic thyroid gland were included in the study. After discontinuation of therapy, a partial or complete organification defect was shown after ¹²³I scintigraphy and perchlorate test.TPO, DUOX2, and DUOXA2 genes were analyzed, and functional activity of DUOX2 variants was studied in HeLa cells.Sequencing of the DUOX2 gene revealed a deletion S965fsX994 in three children; two were euthyroid after 1 month of L-T₄ discontinuation but developed SH after 5 and 18 months, respectively, whereas the other child had SH. One child with SH showed H678R, R701Q, and P982A substitutions, and another child with SH showed only the P982A. One child with SH showed the Y1150C mutation, and another euthyroid child showed the A728T mutation. Functional studies confirmed that S965fsX994, Y1150C, and A728T mutations were responsible for the defect in H₂O₂ production, whereas H678R, R701Q, and P982A did not alter H₂O₂ production in vitro.Genetic analysis of the DUOX2 gene was performed in 11 children with organification defect. Two new mutations (Y1150C and A728T) and the deletion S965FsX994 were responsible for the deficit in the organification process and the phenotypes. Three polymorphisms (H678R, P982A, and R701Q) were identified.

Summary Objective To analyse the coding region of PAX8 in individuals with congenital (CH) or post neonatal hypothyroidism due to dysgenetic (TD) or eutopic thyroid glands. Design and patients Forty-three children with CH and TD (13 agenesis, 23 ectopia, and seven hypoplasia), one subject with post neonatal onset of hypothyroidism and thyroid ectopia, 15 children with CH and eutopic thyroid glands and six euthyroid adults with thyroid hemiagenesis were enrolled as cases, along with 120 healthy individuals as controls. Measurements Exons 2–8 of the PAX8 were directly sequenced. HeLa and HEK293 cells were transfected with PAX8 wild-type (PAX8-WT), mutant PAX8, p300, thyroid transcription factor 1 (TTF-1) and thyroglobulin promoter pGL3 (TG prom-pGL3). Synergism of TTF-1 with PAX8-WT vs. mutant and activity of PAX8-WT vs. mutant in accompaniment with p300 on TG prom-pGL3 were also assessed. The luminescence produced by PAX8-WT and mutant PAX8 was measured. Results Among patients and controls only a 15-year-old girl with thyroid ectopia showed a heterozygous transition of cytosine to thymine at position 674 in exon 6, which changed a conserved threonine at position 225 to methionine (PAX8-T225M). Her father and sister harboured PAX8-T225M without abnormal thyroid phenotypes. PAX8-T225M and PAX8-WT similarly increased luciferase activity and had a similar synergistic effect with TTF-1. At 500 ng p300, however, PAX8-T225M could not significantly increase TG promoter activity when compared to PAX8-T225M alone, while PAX8-WT +500 ng p300 induction was significantly higher than PAX8-WT alone (P

The glycoprotein hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyrotropin (TSH) show low-level cross-reactivity between their respective receptors (R). Patient serum autoantibodies to the thyrotropin receptor (TSHR) do not appear to cross-react with the luteinizing hormone receptor (LHR) or follicle-stimulating hormone receptor (FSHR), although the concentrations of autoantibody with which it is feasible to carry out experiments of this type are limited. Consequently, we have studied the effects of high doses of the thyroid-stimulating human monoclonal autoantibody (M22) on the LHR and FSHR.Chinese Hamster ovary (CHO) cells stably expressing the TSHR, LHR, and FSHR and purified M22 IgG preparations were used in the study.CHO-TSHR, CHO-LHR, and CHO-FSHR cells were incubated with bovine TSH (0.1-25mU/mL), human recombinant chorionic gonadotropin (hCG; 0.5-10mU/mL) or human recombinant FSH (100-5000mU/mL) or with M22 IgG (0.001-5.0 microg/mL), and the extracellular cyclic AMP was measured by radioimmunoassay.Cyclic AMP levels increased in a dose-dependent manner after incubation of CHO-TSHR cells with TSH or M22 IgG, and on a molar basis the effects of TSH and M22 were similar. Cyclic AMP stimulation was not detectable in CHO-LHR and CHO-FSHR cells after incubation with M22 IgG, whereas incubation with hCG or FSH, respectively, caused dose-dependent cyclic AMP stimulation. On a molar basis, concentrations of M22 IgG approximately 100x those of FSH causing clear stimulation were ineffective with CHO-FSHR cells. Similarly, molar concentration of M22 IgG 20,000x those of hCG causing clear stimulation had no effect on CHO-LHR cells.This study shows that at relatively high concentrations, M22 IgG is unable to stimulate cyclic AMP levels in CHO-LHR or CHO-FSHR cells, suggesting that TSHR autoantibodies have greater specificity for the TSHR than TSH itself.

Background: Congenital hypothyroidism is often secondary to thyroid dysgenesis, including thyroid agenesis, hypoplasia, ectopic thyroid tissue or cysts. Loss of function mutations in TSHR, PAX8, NKX2.1, NKX2.5 and FOXE1 genes are responsible for some forms of inherited congenital hypothyroidism, with or without hypoplastic thyroid. The aim of this study was to analyse the PAX8 gene sequence in several members of the same family in order to understand whether the variable phenotypic expression, ranging from congenital hypothyroidism with thyroid hypoplasia to mild subclinical hypothyroidism, could be associated to the genetic variant in the PAX8 gene, detected in the proband. Methods: We screened a hypothyroid child with thyroid hypoplasia for mutations in PAX8, TSHR, NKX2.1, NKX2.5 and FOXE1 genes. We studied the inheritance of the new variant R133W detected in the PAX8 gene in the proband’s family, and we looked for the same substitution in 115 Caucasian European subjects and in 26 hypothyroid children. Functional studies were performed to assess the in vitro effect of the newly identified PAX8 gene variant. Results: A new heterozygous nucleotide substitution was detected in the PAX8 DNA-binding motif (c.397C/T, R133W) in the proband, affected by congenital hypothyroidism with thyroid hypoplasia, in his older sister, displaying a subclinical hypothyroidism associated with thyroid hypoplasia and thyroid nodules, in his father, affected by hypothyroidism with thyroid hypoplasia and thyroid nodules, and his first cousin as well, who revealed only a subclinical hypothyroidism. Functional studies of R133W-PAX8 in the HEK293 cells showed activation of the TG promoter comparable to the wild-type PAX8. Conclusions: In vitro data do not prove that R133W-PAX8 is directly involved in the development of the thyroid phenotypes reported for family members carrying the substitution. However, it is reasonable to conceive that, in the cases of transcriptions factors, such as Pax8, which establish several interactions in different protein complexes, genetic variants could have an impact in vivo.

This study was designed to assess the relationship between mutations in the FSH receptor (FSHr) gene and polycystic ovary syndrome (PCOS) in Italian women. The study population included 50 patients with PCOS and 50 age- and body mass index (BMI)-matched controls. A complete anthropometrical, hormonal and pelvic ultrasonographic evaluation was performed in all subjects. Genomic DNA was extracted from peripheral lymphocytes and then each exon of the FSHr gene was amplified by PCR. The mutation identified was cloned and the functional properties were studied after transient expression in COS-7 cells. Direct sequencing of exons 1–10 of the FSHr gene revealed the presence of a heterozygous AAT/ATT mutation affecting the isoleucine residue at position 411, which was replaced by an asparagine, in the second transmembrane segment (I411N). This mutation was only found in one woman with PCOS and not in her parents. This mutation was not present in 50 age and BMI controls and in another 150 women not affected by PCOS. The functional study after transient expression in COS-7 cells revealed that this I411N had similar functional characteristics with respect to the wild type FSHr (wtFSHr). Genetic analyses of polymorphisms in the human FSHr gene were also performed. All 50 women with PCOS harbored the A307T polymorphic variant, 56% harbored N680S, 30% S680S and 14% N680N polymorphisms. In conclusion, the present study demonstrates that mutations of the FSHr gene are rare in Italian women. The only mutation that we found does not appear to have any pathophysiological significance in PCOS.

Iodine deficiency is the result of insufficient intake of dietary iodine and as a consequence causes multiple adverse effects. About 2 billion individuals in the world are affected by iodine deficiency. It has been found that the most effective way to control iodine deficiency is through the universal salt iodization. However, salt iodization alone may not be sufficient to assure adequate iodine nutrition. In most industrialized countries, excess consumption of salt has become recognized as a health risk. Therefore, biofortification of vegetables with iodine offers an excellent opportunity to increase iodine intake.The aim of this study was to test the efficiency of a new model of iodine prophylaxis in a group of 50 healthy volunteers through the intake of vegetables (potatoes, cherry tomatoes, carrots, and green salad) fortified with iodine. Each serving of vegetables consisted of 100 g of potatoes, carrots, tomatoes, or salad containing 45 mg of iodine (30% of the Recommended Daily Allowance), and the volunteers consumed a single serving of vegetables, as preferred, each day for 2 weeks. Urinary iodine (UI) excretion was measured before and after intake of vegetables.The UI concentration measured in volunteers before the intake of vegetables was 98.3 mg/L (basal value), increasing to 117.5 mg/L during the intake of vegetables. Seven days after the discontinuation of vegetable intake, UI was 85 mg/L. UI concentration increment was 19.6% compared with the basal value; therefore, the difference was statistically significant (P = .035).Biofortification of vegetables with iodine provides a mild but significative increase in UI concentration and, together with the habitual use of iodized salt, may contribute to improve the iodine nutritional status of the population without risks of iodine excess.

Objective Premature ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea) or premature depletion of ovarian follicles before the age of 40 (secondary amenorrhea) with hypergonadotropism and hypoestrogenism. Methods We studied the clinical, biological, and genetic data related to 50 POI patients with a mean age of menopause of 29 years (94% with secondary amenorrhea, 6% with primary amenorrhea and 15% with a family history of POI). Seventeen patients were affected by endocrine autoimmune diseases, antral follicles were observed in 31 patients by ultrasonography. Results Karyotype analysis did not show any abnormality of the X chromosome. No mutation in FSH receptor and GDF-9 genes was reported, while in one patient a variant of BMP-15 gene (A180T) was found. Four patients had fragile X mental retardation 1 gene (FMR1) premutation and one an intermediate sized CGG repeats of the same gene. Two patients with FMR1 premutation were sister and developed secondary amenorrhea at the age of 34 and 37 years. The other two patients presented with oligoamenorrhea at the age of 39 and 34 years. The patient harboured the intermediate sized CGG repeats developed secondary amenorrhea at the age of 33 years. Conclusions The genetic analysis performed on a cohort of patients with POI revealed that 8% had FMR1 premutation and only one patient a previously known variant of BMP-15 gene. No alteration of the karyotype and FSH receptor and GDF-9 genes was evidenced.

ObjectiveMicroRNAs (miRNAs) are small endogenous noncoding RNAs that pair with target messengers regulating gene expression. Changes in miRNA levels occur in thyroid cancer. Fine-needle aspiration (FNA) with cytological evaluation is the most reliable tool for malignancy prediction in thyroid nodules, but cytological diagnosis remains undetermined for 20% of nodules.DesignIn this study, we evaluated the expression of seven miRNAs in benign nodules, papillary thyroid carcinomas (PTCs), and undetermined nodules at FNA.MethodsThe prospective study included 141 samples obtained by FNA of thyroid nodules from 138 patients. miRNA expression was evaluated by quantitative RT-PCR and statistical analysis of data was performed. Genetic analysis of codon 600 of BRAF gene was also performed.ResultsUsing data mining techniques, we obtained a criterion to classify a nodule as benign or malignant on the basis of miRNA expression. The decision model based on the expression of miR-146b, miR-155, and miR-221 was valid for 86/88 nodules with determined cytology (97.73%), and adopting cross-validation techniques we obtained a reliability of 78.41%. The prediction was valid for 31/53 undetermined nodules with 16 false-positive and six false-negative predictions. The mutated form V600E of BRAF gene was demonstrated in 19/43 PTCs and in 1/53 undetermined nodules.ConclusionsThe expression profiles of three miRNAs allowed us to distinguish benign from PTC starting from FNA. When the assay was applied to discriminate thyroid nodules with undetermined cytology, a low sensitivity and specificity despite the low number of false-negative predictions was obtained, limiting the practical interest of the method.

Background: Electric and magnetic fields (EMF) might be involved in human disease and numerous research and scientific reviews have been conducted to address this question. In particular thyroid structural and functional alterations caused by various forms of non-ionizing radiation have been described. Aim: The aim of this study was to analyze the possible effects of EMF on thyroid, in particular we analyzed the effects caused by a GSM (Global System for Mobile Communications) signal (900 MHz) on cultured thyroid cells (FRTL-5). Material and methods: The experimental setup was designed in order to expose samples to a radio frequency wave in well-controlled conditions. We used the FRTL-5 cell line, an epithelial monoclonal continuous cell line derived from Fisher rat thyroid tissue growing as monolayer, expressing the TSH receptor and the sodium-iodide symporter (NIS). FRTL-5 were subsequently irradiate for 24, 48, and 96 h with EMF (800–900 MHz, power-frequency of mobile communication systems) and iodide uptake and cAMP production were measured. Results: The irradiation of cells with EMF at 900 Mhz for 24, 48, and 96 h did not influence the level of cAMP production and was not able to modify iodide accumulation in FRTL-5 cells with respect to basal conditions. Conclusions: In conclusion, EMF do not seem to be able to interfere with the biochemical properties of FRTL-5 cells in vitro.

Background: Thyroid gland is highly dependent on dietary intake of iodine for normal function, so it is particularly subjected to “endocrine disruptor” action. The human sodium/iodide symporter (hNIS) is an integral plasma membrane glycoprotein mediating the active transport of iodide into thyroid follicular cells, a crucial step for thyroid hormone biosynthesis. Beyond to perchlorate and thyocianate ions a few other inhibitors of iodide uptake have been described. Aim: The aim of this study was to investigate if 10 substances usually used as drugs in clinical practice were able to inhibit NIS-mediated iodide uptake in vitro. Materials and methods: A CHO cell line stably expressing hNIS was used to test any inhibition of NIS-mediated iodide uptake exerted by drugs. Perchlorate and thyocianate ions were used as positive controls. Results: None of the analyzed substances was able to significantly inhibit iodide uptake in our system. As we expected, perchlorate and thyocianate ions were able to inhibit iodide uptake in a dose-dependent manner. Conclusions: In conclusion, we carried out an in vitro assay to evaluate the potential inhibitory effect of common drugs on NIS-mediated iodide uptake by using CHO-hNIS cells. None of the analyzed substances was able to inhibit iodide uptake; only perchlorate and thyocianate were able to inhibit iodide uptake in a dose-dependent manner.

Background: Vitiligo is an acquired depigmenting disorder characterized by the loss of melanocytes from the epidermis with the development of white patches in various distribution. The pathogenesis of vitiligo is still unknown, but the association with autoimmune disorders and organ specific autoantibodies, supports the hypothesis of an autoimmune pathogenesis. Aim: The aim of the present study was to investigate if autoantibodies present in sera of patients affected by vitiligo may be able to interfere with the activity of the αMSH on the melanocortin 1 receptor (MC1R). Materials/Subjects and methods: IgG from the sera of 41 patients with vitiligo associated or not with thyroid autoimmune diseases or other autoimmune pathologies were incubated with HBL20 cells (human malignant melanocytes expressing the MC1R) in the presence of a sub-maximal dose of αMSH. A normal IgG range was determined by using IgG extracted from 30 control sera of normal subjects. Results: None of the IgG from vitiligo patients was able to inhibit αMSH-stimulated cAMP production in HBL20 cells. Conclusions: Autoantibodies against MC1R are rare or absent in sera of vitiligo patients.

In a previous work we found that the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), inhibits the accumulation of cAMP as induced by the bovine thyroid stimulating hormone (bTSH) in cells transfected with the TSH receptor. In this work, we demonstrate that the DDT molecular analogues, diethylstilbestrol and quercetine, are more potent inhibitors of the TSH receptor activity than DDT itself. The notion that all these compounds interfere with nuclear estrogen receptors, as either agonists (DDT and diethylstilbestrol) or antagonists (quercetin), prompted us to test the ability of the steroid hormone 17-beta-estradiol to inhibit the TSH receptor activity. We found that estrogen exposure causes a modest but significant inhibition of the bTSH induced cAMP accumulation both in transfected CHO-TSH receptor and Fischer Rat Thyroid Low Serum 5% (FRTL-5) cells. When applied to CHO cells transfected with the luteinizing hormone receptor, 17-beta-estradiol proved capable of inhibiting the hCG induced cAMP accumulation at a concentration as low as 10nM, though the effect was not greater than 35%. The effect of 17-beta-estradiol was not estrogen receptors mediated, as co-transfection of the estrogen receptor alpha and beta subunits with LH receptor caused cAMP to increase above the level attained by the sole hCG stimulation, and not to decrease it as expected. These data suggest the presence of a steroidal-like allosteric binding site on glycoprotein hormone receptors.

Some cases of congenital hypothyroidism (CH) are associated with a gland of normal size.To explore the cause of organification defect in one child with CH and a eutopic thyroid gland, genetic analyses of TPO, DUOX2, and DUOXA2 genes were performed.One child with CH, a eutopic thyroid gland, and a partial organification defect was shown after (123)I scintigraphy and perchlorate test.In the child with the organification defect, TPO, DUOX2, and DUOXA2 genes were analyzed. The functional activity of the DUOX2 mutants was studied after expression in eukaryotic cells.No TPO or DUOXA2 gene mutations were identified. Direct sequencing of the DUOX2 gene revealed a compound heterozygous genotype for S911L and C1052Y substitutions. S911L and C1052Y caused a partial defect in H(2)O(2) production after transient expression in HeLa cells.We performed a genetic analysis in one child with CH and a eutopic thyroid gland. Two new mutations in DUOX2 gene responsible for the partial deficit in the organification process were identified.

Loss-of-function mutations of the thyrotropin receptor (TSHr) may be responsible for congenital hypothyroidism or isolated hyperthyreotropinemia. To study cell surface expression of inactivating TSHr mutations detected in patients with isolated hyperthyreotropinemia (L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T), we used the Agilent 2100 bioanalyzer to perform microchip flow cytometry analysis. The previously described TSHr inactivating mutation T477I was used as control. The level of receptor expression in COS-7 cells transfected with the T477I measured by binding assay was four times lower with respect to the wild-type TSHr. The very low expression of T477I was confirmed by fluorescence-activated cell sorting (FACS) analysis and by microchip flow cytometry analysis, suggesting that this method can be a reliable system to measure receptor cell surface expression. Other inactivating TSHr mutations were expressed in COS-7 cells for binding studies, FACS analysis, and microchip flow cytometry analysis. Binding studies showed that L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T mutants had a low expression at the cell surface, as demonstrated by Bmax values. Data obtained by binding studies were in good agreement with data obtained by FACS analysis and microchip flow cytometry analysis. In conclusion, the low number of cells required for analysis and the ease of use make the microchip flow cytometry analysis a very reliable and favorable system to study cell surface expression of TSHr mutations.

Summary Background Iodide (I−) is crucial for foetal thyroid function. Foetal iodide results from maternal circulating iodide and from deiodination of iodothyronines within the placenta. The Na+/I− symporter (NIS) localized in placental cells appears to be involved in iodide exchange. Low NIS expression has been reported in trophoblast cells from the first trimester and pregnancy at term. Aims The aim of this study was to examine NIS expression by immunohistochemistry in the major components of human ovular tissue and placenta. Materials and methods Formalin-fixed and paraffin-embedded specimens of placental tissue from the first trimester and at term were analysed. NIS expression was quantified as percentage of NIS-positive cells/total cells. NIS expression was also evaluated by real-time polymerase chain reaction (RT-PCR) in five first-trimester and five at-term placental specimens. Results In the first-trimester specimens heterogeneous NIS immunoreactivity was found in cyto-syncytiotrophoblast cells, with a range of NIS-positive cells from 5% to 80% (mean ± SD 21·85 ± 23·95), in mesenchymal and endothelial cells from 1% to 40% (14·5 ± 11·16), in decidual cells from 5% to 40% (10·38 ± 11·98) and in endometrial glands from 3% to 40% (21·86 ± 13·93). In specimens from placenta at term, NIS-positive cyto-syncytiotrophoblast cells were between 5% and 40% (mean 17·85 ± 18·15), mesenchymal and endothelial cells between 1% and 40% (13·67 ± 12·16), decidual tissue between 5% and 30% (16·43 ± 9·08), and endometrial glands between 3% and 40% (16·67 ± 15·27). No significant differences in NIS expression were observed between the first trimester and placenta at term. A similar level of mRNA expression for the NIS gene was obtained by RT-PCR both in ovular material of the first trimester and in placenta at term. Conclusions We found NIS to be expressed in various placental and ovular components and its expression to remain constant during pregnancy.

Summary background Premature ovarian failure (POF) is defined by cessation of ovarian function after puberty and before the age of 40. The syndrome is characterized by amenorrhoea, oestrogen deficiency and elevated levels of gonadotrophins. Autoimmunity has been proposed as a mechanism for some cases of destruction or malfunction of ovarian follicles. POF is often associated with type I and type II polyglandular autoimmune syndromes. It has also been postulated that receptors such as the LH and FSH receptors might become targets for blocking antibodies and such antibodies could be a cause of ovarian failure. patients and methods Sixty-nine patients with POF isolated or associated with other endocrine autoimmune diseases (autoimmune thyroid diseases, Addison's disease, type 1 diabetes mellitus, multiple sclerosis, myasthenia gravis) were studied. All the patients had secondary amenorrhoea. The patient group had a median age of 33·1 years (range 15–57). Ovarian failure had been diagnosed at a median age of 29 years (range 15–39). The median time since diagnosis was almost 1 year but in six patients gonadal insufficiency had appeared 10–30 years earlier. All had a normal chromosomal karyotype (46, XX). Patients with POF were characterized by duration of amenorrhoea > 1 year, with elevated FSH and LH levels and undetectable or low oestrogen levels. Cell lines stably expressing recombinant human LH (CHO-LHr) and FSH (CHO-FSHr) receptors were prepared and used to search for antibodies able to inhibit LH- or FSH-stimulated cAMP production. Immunoglobulins extracted from sera of patients with POF were incubated with CHO-LHr and CHO-FSHr in the presence of human recombinant CG and FSH, respectively. results and conclusions None of the immunoglobulin G (IgG) preparations from patients with POF was able to inhibit the activity of the FSH- and CG-stimulated cAMP production.

OBJECTIVE: TSH receptor (TSHr) mediates the activating action of TSH on the thyroid gland resulting in the growth and proliferation of thyrocytes and thyroid hormone production. TSHr is a major autoantigen in Graves' disease (GD) and is the target for TSHr antibodies. In GD, thyroid-stimulating antibodies (TSAb) are competitive agonists of TSH. In atrophic thyroiditis (AT), thyroid-stimulating blocking antibodies (TSHBAb) are TSH antagonists. The TSHr together with the LH receptor (LHr) and FSH receptor (FSHr) are G-protein-coupled receptors with considerable amino acid homologies in the extracellular domain. We studied the cross-reactivity of the antibodies measured in sera from patients with GD or AT on the LHr and FSHr function. METHODS: We tested the activity of TSAb and TSHBAb in cell lines expressing the LHr and the FSHr. To this purpose a pSVL-FSHr construct was transfected in CHO cells and one clone was used. RESULTS: Twenty-eight sera from patients with GD and four from patients with AT, known to contain TSHr antibodies measured with a radioreceptor assay, were selected. TSAb and TSHBAb activities were measured in CHO cells expressing the TSHr (CHO-TSHr). TSAb and TSHBAb were then tested with the cell lines expressing the LHr and the FSHr for their ability to elicit cAMP accumulation or inhibit FSH/LH-induced cAMP production. None of the TSAb identified was able to stimulate cAMP increase in CHO-LHr or CHO-FSHr. Similarly, none of the TSHBAb was able to block the cAMP response induced by FSH or LH in the respective cell lines. CONCLUSIONS: Our results confirm the notion of the organ-specific nature of the TSHr antibodies.

The presence of perchlorate (ClO(4) (-)) in some U.S. drinking water supplies has raised concern about potential adverse thyroidal health effects, because ClO(4) (-) is known to competitively inhibit iodide uptake at the sodium iodide symporter (NIS). Humans are nutritionally and environmentally exposed to other competitive inhibitors of iodide uptake, including thiocyanate (SCN(-)) and nitrate (NO(3) (-)). The joint inhibiting effects of these three anions was studied by exposing Chinese hamster ovary cells stably expressing human NIS to varying concentrations of each anion separately, and in combination, and conducting measurements of (125)I(-) uptake. The entire data set was fit to a single Hill equation using maximum likelihood. The relative potency of ClO(4) (-) to inhibit (125)I(-) uptake at the NIS was found to be 15, 30 and 240 times that of SCN(-), I(-), and NO(3) (-) respectively on a molar concentration basis, with no evidence of synergism. These results are consistent with a common mode of action by these anions of simple competitive interaction, in which a concentration of any one of ClO(4) (-) SCN(-), and NO(3) (-), occurring either individually or as part of a mixture of the three anions, is indistinguishable from a concentration or dilution of either one of the remaining two ions in inhibiting iodine uptake at the NIS.

Riassunto Scopo principale di questo lavoro e l'illustrazione per mezzo del microscopio a scansione, delle spore delle Pteridofite spontanee in Italia. Queste ammontano a 124 taxa specifici ed infraspecifici, le cui spore sono illustrate da 550 micrografie (3–8 per ogni taxon) radunate in 71 tavole. Il lavoro consiste di una prefazione, una introduzione e quattro parti del testo vero e proprio. La prefazione e principalmente destinata a spiegare lo scopo del lavoro ed a ringraziare gli amici e colleghi che ci hanno offerto gentilmente aiuto nella nostra ricerca. L'introduzione e rivolta a dare alcune informazioni generali e dettagli sull'inquadramento tassonomico delle Pteridofite italiane, delle quali viene dato un elenco completo, sui materiali e metodi per il nostro studio, e sulle ricerche per reperire i dati citologici e geobotanici. La prima parte fornisce i dati relativi a ciascuna specie e sottospecie, eccezionalmente varieta, studiate nel nostro lavoro. Essi consistono di varie informazioni suddi...

Riassunto L'analisi corologica di alcune ombrellifere rare delle Alpi Apuane, mette in evidenza che Trinia dalechampii (Ten.) Janchen e Carum apptianum (Viv.) Grande e specie affini (Carum heldreichii Boiss. e C. multiflorum (S. et S.) Boiss. sono distribuite sporadicamente in relitti sui resti della catena che nell'era cenozoica, nell'Oligocene, emergeva dal mare dall'Asia minore alla Grecia e all'Appennino; catena che nel Pontico dalla Grecia mandava un ramo lungo le coste adriatiche fino alle Alpi orientali (FERRARINI, 1965 fig. 5). Athamanta con A. macedonica (L.) Sprengel, A. sicula L., A. cortiana Ferrarini risale la penisola italiana fino alle Alpi Apuane, con A. turbith (L.) Brot, la Penisola balcanica fino alle Alpi Giulie e inoltre con A. sicula L. si spinge, attraverso la Sicilia, fino all'Algeria. Trochiscanthes nodiflora (Vili.) Koch e Astrantia minor L. hanno un areale che gravita attorno alle Alpi occidentali. Trochiscanthes nodiflora ha stazioni nell'Aude in Francia e nel Trentino che potr...