Bernard Rousset

Evidence for Transcriptional and Posttranscriptional Alterations of the Sodium/Iodide Symporter Expression in Hypofunctioning Benign and Malignant Thyroid Tumors

The uptake of iodide by epithelial thyroid cells requires the expression of a specific transporter, the Na(+)/I(-) symporter, NIS. Benign and malignant thyroid tumors of epithelial origin show a decrease up to a loss of iodide uptake activity. Previous studies of the human NIS (hNIS) gene expression in these tumors, based on the amplification of transcripts and/or immunohistochemical detection of the protein, have yielded divergent data; hNIS expression was found either increased or decreased. To get a new and integrated view of the alterations of hNIS expression in hypofunctioning thyroid tumors, we performed investigations of hNIS transcript and hNIS protein levels on the same tumors and paired normal tissue samples. HNIS, identified as a 75- to 80-kd species, was present in all normal tissue samples from euthyroid patients, but was undetectable, even at high membrane protein input, in all benign and malignant hypofunctioning thyroid tumors. By contrast, approximately 50% of tumors contained hNIS transcripts. This dissociation between transcript and protein levels was not found for the transcript and protein encoded by the PDS gene assayed in the same tumors. The hNIS transcript-positive tumors contained small amounts of low-molecular mass hNIS-immunoreactive species identified as nonglycosylated hNIS. Tumors containing the nonmature form of hNIS exhibited a predominant intracellular immunolabeling. In conclusion, our data show that benign and malignant hypofunctioning thyroid tumors either no longer express hNIS protein or express only a very low amount of nonglycosylated hNIS and indicate that the impairment of hNIS gene expression might result from alterations at both transcriptional and posttranscriptional levels.

Oncogenic alterations in papillary thyroid cancers of young patients.

BACKGROUNDnPapillary thyroid carcinoma (PTC) in young people usually has an aggressive initial presentation, though a good general prognosis despite recurrences in 10%-20% of patients. A number of genetic alterations that activate the mitogen-activated protein kinase (MAPK) pathway have been found in PTC. Some of these alterations have been identified as prognostic factors of PTC in adults. The objective of the current study was to comprehensively characterize all known oncogenic alterations of the MAPK pathway in young people.nnnMETHODSnOne hundred three PTCs removed from 9 children, 19 adolescents, and 75 young adults were submitted to molecular analyses.nnnRESULTSnAltogether, 57 alterations were found in 56 PTCs (55%) corresponding to V600E BRAF in 20.3%, RAS mutations in 12.6%, RET/PTC 1 in 11.6%, RET/PTC 3 in 8.7%, and rearrangement of NTRK in 1.9%. The prevalence of all alterations increased with age (22.2% in children; 52.6% in adolescents, 51.4% in adults 20-25 years, and 55.1% in adults 25-35 years). Prevalence increased from 39.2% earlier to 61.3% after 20 years mainly due to BRAF mutations. Classic-type PTC was associated with a larger prevalence of alterations, predominantly BRAF and RET/PTC, whereas the follicular variant was chiefly associated with RAS. RET/PTC (1 and 3) was significantly associated with extrathyroid extension (ET) and lymph node metastasis (es) (LNM). This association was found in the adult group. There were no associations of BRAF or RAS mutations with ET or LNM. A 3-year median follow up was available for 90 patients. RET/PTC 1 and 3 was associated with short-term disease dissemination (cervical lymph node recurrences and distant metastases) in young adults (p=0.001). Persistent illness was more prevalent in patients with (15%) than in patients without (7%) genetic alterations.nnnCONCLUSIONnPTCs in young patients display a low prevalence of the already identified oncogenic alterations. The increasing prevalence with age is mainly due to V600E BRAF mutation. There is no relation between tumor aggressiveness and BRAF mutation. There is a relation between the presence of RET/PTC (1 and 3) and the histological and clinical short-term aggressiveness of PTC in the population of young adults. Such a relation is not found in children and adolescents.

Evaluation of Gene Expression Profiles in Thyroid Nodule Biopsy Material to Diagnose Thyroid Cancer

CONTEXTnDetection of thyroid cancer among benign nodules on fine-needle aspiration biopsies (FNAB), which presently relies on cytological examination, is expected to be improved by new diagnostic tests set up from genomic data.nnnOBJECTIVEnThe aim of the study was to use a set of genes discriminating benign from malignant tumors, on the basis of their expression levels, to build tumor classifiers and evaluate their capacity to predict malignancy on FNAB.nnnDESIGNnWe analyzed the level of expression of 200 potentially informative genes in 56 thyroid tissue samples (benign or malignant tumors and paired normal tissue) using nylon macroarrays. Gene expression data were subjected to a weighted voting algorithm to generate tumor classifiers. The performances of the classifiers were evaluated on a series of 26 sham FNAB, i.e. FNAB carried out on thyroid nodules after surgical resection.nnnRESULTSnA series of 19 genes with a similar expression in follicular adenomas and normal tissue and discriminating follicular adenomas+normal tissue from the following: 1) follicular thyroid carcinomas (FTCs), 2) papillary thyroid carcinomas (PTCs), or 3) both FTCs and PTCs. These were used to generate four classifiers, the FTCs, PTCs, common (FTC+PTCs), and global classifiers. In 23 of the 26 sham FNAB, the four classifiers yielded a diagnosis in agreement with the diagnosis of the pathologist used as reference; in the three other cases, the correct diagnosis was given by three of four classifiers.nnnCONCLUSIONSnWe developed a procedure of molecular diagnosis of benign vs. malignant tumors applicable to the material collected by FNAB. The molecular test complied with a preclinical validation stage; it must be now evaluated on ultrasound-guided FNAB in a large-scale prospective study.

Molecular characteristics of papillary thyroid carcinomas without BRAF mutation or RET/PTC rearrangement: relationship with clinico-pathological features.

About 60-70% of papillary thyroid carcinomas (PTC) present a BRAF(T1799A) gene mutation or a rearrangement of RET gene (RET/PTC). In this study, we examined whether PTC without BRAF(T1799A) mutation and without RET/PTC rearrangement named PTC-ga(-) were distinguishable from PTC-ga(+) (with one or the other gene alteration) on the basis of gene expression characteristics. We analyzed the mutational state of 116 PTC and we compared gene expression profiles of PTC-ga(+) and PTC-ga(-) from data of a 200 gene macroarray and quantitative PCR. Seventy five PTC were PTC-ga(+) and 41 were PTC-ga(-). Unsupervised analyses of macroarray data by hierarchical clustering led to a complete segregation of PTC-ga(+) and PTC-ga(-). In a series of 42 genes previously recognized as PTC marker genes, 22 were found to be expressed at a comparable level in PTC-ga(-) and normal tissue. Thyroid-specific genes, TPO, TG, DIO1, and DIO2 were under-expressed in PTC-ga(+) but expressed at a normal level in PTC-ga(-). A few genes including DUOX1 and DUOX2 were selectively dys-regulated in PTC-ga(-). Tumor grade of PTC-ga(-) was lower than that of PTC-ga(+). There was a strong association between the mutational state and histiotype of PTC; 81% of PTC follicular variants were corresponded to PTC-ga(-), whereas 84% of PTC of classical form were PTC-ga(+). In conclusion, we show that PTC without BRAF(T1799A) mutation or RET/PTC rearrangement, mainly corresponding to follicular variants, maintain a thyroid differentiation expression level close to that of normal tissue and should be of better prognosis than PTC with one or the other gene alteration.

Melanin concentrating hormone in central hypersomnia

BACKGROUNDnNarcolepsy with cataplexy (NC) is a disabling disorder characterized by excessive daytime sleepiness and abnormal rapid eye movement (REM) sleep manifestations, due to a deficient hypocretin/orexin neurotransmission. Melanin concentrating hormone (MCH) neurons involved in the homeostatic regulation of REM sleep are intact. We hypothesized that an increased release of MCH in NC would be partly responsible for the abnormal REM sleep manifestations.nnnMETHODSnTwenty-two untreated patients affected with central hypersomnia were included: 14 NC, six idiopathic hypersomnia with long sleep time, and two post-traumatic hypersomnia. Fourteen neurological patients without any sleep disorders were included as controls. Using radioimmunoassays, we measured hypocretin-1 and MCH levels in cerebrospinal fluid (CSF).nnnRESULTSnThe MCH level was slightly but significantly lower in patients with hypersomnia (98 ± 32 pg/ml) compared to controls (118 ± 20 pg/ml). After exclusion of patients affected with post-traumatic hypersomnia the difference became non-significant. We also failed to find any association between MCH level and hypocretin level, the severity of daytime sleepiness, the number of SOREMPs, the frequency of cataplexy, and the presence of hypnagogic hallucinations or sleep paralysis.nnnCONCLUSIONnThis study reports the first measurement of MCH in CSF using radioimmunoassay technology. It appears to be a non-informative tool to differentiate etiologies of central hypersomnia with or without REM sleep dysregulation.

Tubulin-chromatin interactions: evidence for tubulin-binding sites on chromatin and isolated oligonucleosomes.

The interaction of tubulin with chromatin has been studied using a radiolabeled tubulin binding assay and velocity sedimentation analysis on isokinetic sucrose gradients. Soluble chromatin was prepared by mild micrococcal nuclease digestion of rat liver nuclei and tubulin was purified from rat brain by temperature-dependent assembly-disassembly and phosphocellulose chromatography. The tubulin-binding assay is based on the ability of chromatin to precipitate quantitatively at physiological ionic strength allowing separation of free tubulin from chromatin-bound tubulin. The binding of tubulin to unfractionated soluble chromatin was rapid, reversible and saturable. Saturation of binding sites was obtained using tubulin concentrations ranging from 0.5 to 400 micrograms/ml, in the presence of a high concentration (2.5 mg/ml) of another acidic protein, bovine serum albumin. The Scatchard and Hill plots showed that tubulin bound to a single class of non-interacting sites and yielded values of (0.5-0.6) X 10(7) M-1 for an apparent Ka and a maximal binding capacity of 0.8 nmol tubulin/mg DNA, i.e. about 1 molecule of tubulin/10 nucleosomes. Similar binding parameters were obtained when binding experiments were performed with insoluble chromatin in 0.15 M NaCl. Velocity sedimentation analysis of tubulin-chromatin complexes revealed that tubulin bound to all classes of chromatin oligomers, irrespective of the length of the nucleosomal chain. Tubulin-trinucleosome complexes formed from isolated trinucleosome in the presence of an excess of tubulin were separated from free reactants. It was found that 10-15% of the starting oligonucleosomal species reacted with tubulin, in a stoichiometry of about 0.8 molecule of tubulin/nucleosome. Given the characteristics of the binding and the expected cellular free tubulin concentration, the tubulin-chromatin interaction could possibly take place in vivo, when the nuclear membrane breaks down during the first steps of mitosis.

Culture of Dendritic Cells from a Nonlymphoid Organ, the Thyroid Gland: Evidence for TNFα-Dependent Phenotypic Changes of Thyroid-Derived Dendritic Cells

Because they are sparsely distributed in tissues, dendritic cells (DC) present in nonlymphoid organs are difficult to isolate. Only DC from skin and lung have been successfully studied in culture. The objective of the present work was to investigate the possibility of isolating and culturing DC from an endocrine organ, the thyroid gland, which is particularly susceptible to the development of autoimmune processes. The study was conducted on pig thyroid glands to have sufficient amounts of starting material. This choice required the characterization of immunological reagents capable of recognizing DC markers in the pig species. Using a discontinuous trypsinization procedure, a DC population representing 2% to 3% of the thyroid cell suspension was reproducibly obtained. Isolated DC quantitatively attached to tissue culture-treated dishes and segregated from thyrocytes. DC identified as cells expressing major histocompatibility complex class II molecules, the mannose receptor, and the S100 protein were found to have a high capacity to internalize labeled ligands, dextran, and mannosylated albumin. These cells had a phenotype of immature DC. Secondarily, a fraction of DC detached from culture dishes, and floating DC had low or no endocytic activity, a characteristic of mature DC. Treatment of DC/thyrocytes cocultures with tumor necrosis factor α (TNFα) activated the transformation of immature DC into mature DC. These data show that DC isolated from the thyroid gland can be maintained immature or activated to undergo maturation in primary culture. The procedure of cell isolation and culture should be adaptable to human thyroid tissue for in vitro analyses of DC-mediated immune responses.

Variations of rat thyroid activity during exposure to high environmental temperature (34° C)

SummaryChanges in thyroid activity and variations in the hypothalamopituitary-thyroid hormone levels were examined in rats exposed to heat (34° C) for 3 weeks. Thyroid activity evaluated histologically (epithelium/colloid ratio, nuclear size), by radioiodine exploration (24 hrs —125I uptake, ratio of mono- to di-125 iodotyrosines — MIT/DIT, ratio of tri- to tetra-125 iodothyronines-T3/T4, and plasma125I-T4 and assay of plasma T4, evolves in a triphasic manner: 1. a depression phase between day 0 and day 2.5. 2. a rebound of thyroid activity between day 2.5 and day 9. 3. a stabilization of thyroid parameters from day 9 to day 24. These results indicate adaptation of thyroid function to heat after 3 weeks. In phase I, plasma TSH (MeKenzie bioassay) fell to undetectable levels concurrent with a 50% decrease in hypothalamic TRH (in vitro assay). Plasma TSH peaked on day 4.5, fell on day 9.5 and returned progressively to initial levels. Hypothalamic TRH returned to initial levels after 6.5 days.The rapid and simultaneous decrease in hypothalamic TRH, plasma TSH, plasma T4 and thyroid activity by the 36th hour of heat exposure (34° C) suggests initiation at the hypothalamic level. In the second phase, the rebound in thyroid activity is presumably due to the peak in circulating TSH in relation to the marked decrease in plasma T4. The oscillations of phase 2 and the stabilization of all the thyroid parameters in phase 3 may be the reflection of an adaptive process. At the end of this adaptive period an apparent discrepancy remains between a low plasma T4 and a normal or subnormal plasma TSH. A modification in the “set point” for the control of TSH secretion is discussed.

Signaling from epithelial to dendritic cells of the thyroid gland: evidence for thyrocyte-derived factors controlling the survival, multiplication, and endocytic activity of dendritic cells.

Intrathyroidal dendritic cells (DC) isolated at the same time and then cultured with thyrocytes in the presence of thyrotropin (TSH) keep a phenotype of immature DC (Croizet et al, 2000). As DC from other sources are known to undergo a rapid maturation in vitro, we hypothesized that the maintenance of thyroid-derived DC in an immature state might be caused by thyrocytes-DC interactions. In this study, we investigated whether thyroid-derived DC could change their phenotype in response to TSH stimulation of thyrocytes. Over an 8-day period of culture, the population of DC increased 2- to 3-fold in the presence of TSH and decreased by more than 75% in the absence of TSH. The increase in the DC population was related to DC proliferation, whereas the reduction of the number of DC was secondary to a loss of cell-substrate adhesion and subsequent cell death. In the presence of TSH, DC acquired and maintained a high capacity for internalizing labeled ligands, expressed the mannose receptor, and exposed MHC class II molecules at the cell surface. On the contrary, DC cultured without TSH were devoid of endocytic activity and mannose receptor and, after 2 days, no longer exposed MHC class II molecules at the cell surface. Using conditioned media and enriched DC populations, we show that thyrocytes, in response to TSH, produce soluble factors capable of activating proliferation and endocytic activity of DC. Exogenous granulocyte/macrophage-colony stimulating factor and transforming growth factor-β, known to be produced by thyrocytes, reproduced the effects of conditioned media. These data, giving evidence of a hormone-regulated signaling process between epithelial and dendritic cells in vitro, suggest that thyrocytes could promote the maintenance of a population of immature DC within the thyroid gland.

Regulation of TSH secretion in rats chronically exposed to heat (34C)

Previous studies have shown that in heat exposed rats, a decreased plasma T4 concentration was associated with a normal biologically active TSH concentration. This study was designed to clarify this apparent discrepancy in the regulation of TSH secretion. In experimental rats (34°C for 25 days) and controls (25°C), plasma total T4 was 3.2 vs. 5.7×10−8 mol/l. (P< 0.01), plasma total T3: 2.4 vs. 5.7×10−10 mol/l. (P<0.01) and plasma TSH: bioassay 0.34 vs. 0.29 mU/ml (ns), radioimmunoassay: 1.04 vs. 0.87 μg RP1/ml (ns), After TRH, plasma TSH increased identically in the two groups. In heat-exposed rats, the dialysable fraction of T4 and T3 were increased: 0.032 vs. 0.020% (P<0.05) and 0.102 vs. 0.086% (P<0.05), respectively; accordingly, free T4 concentration was normal and that of free T3 was low; total plasma proteins were slightly increased.It is concluded that in heat-exposed rats: (1) plasma thyroid hormone binding activity was decreased as shown by the association: decreased plasma total T4-elevated free T4 fraction. The normality of the free T4 concentration accounted for the normal plasma TSH. (2) the combination of normal plasma TSH, normal plasma free T4, low plasma free T3 concentrations would suggest that T4 is predominantly involved in the regulation of TSH secretion.