Evidence for processing of compact insoluble thyroglobulin globules in relation with follicular cell functional activity in the human and the mouse thyroid.

OBJECTIVE: Thyroglobulin (Tg) is stored within the follicular lumen mainly in a soluble form, but globules made of insoluble multimers are also present and considered to be a mechanism to store prohormone at high concentration. We investigated the immunohistochemical properties of these intrafollicular globules and their possible processing by thyroid cells upon stimulation in the human and in the mouse. DESIGN: Human thyroids (normal, Graves' disease and hot adenomas) and thyroids from old ICR mice without or with goitrogenic treatment were processed for light microscopy. METHODS: Immunohistochemistry for Tg with a polyclonal antibody and two monoclonal antibodies, one specific for thyroxine-rich-iodinated Tg and the other recognizing Tg independently of its iodine level, staining with periodic-acid-schiff, and binding of lectins specific for mannose and sialic acid were performed on all tIssue sections. Intrafollicular globules were quantified, with distinction between 'active' or 'hot' and 'hypofunctioning' or 'cold' follicles. RESULTS: In normal human and old mouse thyroids, the intrafollicular globules were strongly stained with PAS, but negative for the three anti-Tg antibodies and the two lectin-binding assays, while the surrounding soluble Tg was positive. In normal human tIssue, globules were more frequent in 'hypofunctioning' than in 'active' follicles. They were exceptional in Graves' disease and hot adenomas. In old mice, Tg globules were more frequent in 'cold' than in 'hot' follicles. Along with the goitrogen treatment, they became fewer, fragmented and more often present in follicles with a 'hot' aspect. CONCLUSIONS: Upon TSH stimulation, thyrocytes become able to process colloid globules suggesting that this stock of Tg can be used in vivo for thyroid hormone synthesis.

Correlation between the loss of thyroglobulin iodination and the expression of thyroid-specific proteins involved in iodine metabolism in thyroid carcinomas.

Progress in biotechnology has provided useful tools for tracing proteins involved in thyroid hormone synthesis in vivo. Mono- or polyclonal antibodies are now available to detect on histological sections the Na(+)/I(-) symporter (NIS) at the basolateral pole of the cell, the putative iodide channel (pendrin) at the apical plasma membrane, thyroperoxidase (TPO), and members of the NADPH-oxidase family, thyroid oxidase 1 and 2 (ThOXs), part of the H(2)O(2)-generating system. The aim of this study was to correlate thyroglobulin (Tg) iodination with the presence of these proteins. Tg, T(4)-containing Tg, NIS, pendrin, TPO, ThOXs, and TSH receptor (TSHr) were detected by immunohistochemistry on tissue sections of normal thyroids and various benign and malignant thyroid disorders. Tg was present in all cases. T(4)-containing Tg was found in the adenomas, except in Hurthle cell adenomas. It was never detected in carcinomas. NIS was reduced in all types of carcinomas, whereas it was detected in noncancerous tissues. Pendrin was not expressed in carcinomas, except in follicular carcinomas, where weak staining persisted. TPO expression was present in insular, follicular carcinomas and in follicular variants of papillary carcinomas, but in a reduced percentage of cells. It was below the level of detection in papillary carcinomas. The H(2)O(2)-generating system, ThOXs, was found in all carcinomas and was even increased in papillary carcinomas. Its staining was apical in normal thyroids, whereas it was cytoplasmic in carcinomas. The TSHr was expressed in all cases, but the intensity of the staining was decreased in insular carcinomas. In conclusion, our work shows that all types of carcinomas lose the capacity to synthesize T(4)-rich, iodinated Tg. In follicular carcinomas, this might be due to a defect in iodide transport at the basolateral pole of the cell. In papillary carcinomas, this defect seems to be coupled to an altered apical transport of iodide and probably TPO activity. The TSHr persists in virtually all cases.

Relationships between cell division, expression of growth factors and microcirculation in the thyroids of Tg-A2aR transgenic mice and patients with Graves' disease.

Tissue heterogeneity and nodule formation are hallmarks of thyroid growth. This is accounted for by the clonality theory that acknowledges different individual cellular abilities to respond to trophic stimuli. In order to test the hypothesis that functional and mitotic properties of thyrocytes could be influenced by paracrine interactions with neighbour endothelial cells, studies were conducted in both mouse and human goitre models. In the first part of the study, homogenous goitres in C57 black mice were compared with heterogeneous goitres in transgenic hyperthyroid mice expressing the A2 adenosine receptor (Tg-A2aR). The second part of the study concentrated on comparing human thyroid tIssue of control individuals and of patients with Graves' disease. The rate of cell division was evaluated by immunohistochemical detection of cells positive for proliferating cell nuclear antigen (PCNA). Their spatial distribution was then correlated with immunohistochemical cellular expression of growth- and vasoactive-related factors (fibroblast growth factor-2, transforming growth factor-beta, endothelin-1, vascular endothelial growth factor, nitric oxide synthase III), and with microcirculation expansion. Observations were made on digitalised images of histological serial sections. The nearest-neighbour method was used to distinguish between random or clustered distribution. PCNA-positive cells were both randomly and uniformly distributed in homogenous goitres from C57 black mice, and were clustered in tIssue areas identified as papillary and hyperplastic zones in heterogeneous goitres from Tg-A2aR mice. However, they were absent in the so-called compact cellular zones featuring resting cells. Moreover, whereas papillary and hyperplastic zones were highly vascularised, compact zones were nearly free of microvessels. Spatial distribution of dividing cells was positively correlated with the expression of growth-related factors. A similar pattern was observed in the thyroids of patients with Graves' disease. In accordance with the recent demonstration of the presence of angiofollicular units in the thyroid, these data strongly support the hypothesis that functional and mitotic properties of each single thyrocyte, likely to be responsible for growth heterogeneity of hyperplastic glands, may be adjusted at tIssue level by specific interactions with neighbour endothelial cells that, in turn, could alter the mitotic rate of thyrocytes through paracrine signals.

Structural changes in the angiofollicular units between active and hypofunctioning follicles align with differences in the epithelial expression of newly discovered proteins involved in iodine transport and organification.

In animals, as well as in humans, the thyroid gland is made of active follicles, with cuboidal cells and hypofunctioning follicles, with flattened cells. In this study, the functional status of human follicles was dissected out, based on immunohistochemical detection of TSH receptor, Na(+)/I(-) symporter, pendrin, thyroperoxidase (TPO), thyroid oxidases (ThOXs), and T(4)-containing iodinated Tg (Tg-I). To ascertain that angiofollicular units exist in the human, we studied the microvascular bed of each follicle, in correlation with detection of vascular endothelial growth factor (VEGF), of nitric oxide synthase III, and of endothelin in normal and goitrous thyroids. In hypofunctioning follicles, pendrin, TPO, and ThOXs were not detected, and there was no Tg-I in the colloid. At the opposite, in active follicles, pendrin, TPO, and ThOXs were detected in thyrocytes, and Tg-I was present in the colloid. In normal and goitrous thyroids, the capillary networks surrounding active follicles were larger than those surrounding hypofunctioning follicles. Immunoreactivity for nitric oxide synthase III and endothelin was solely detected in active follicles. Only a few follicles in normal thyroids were immunostained for VEGF, regardless of their functional status. In multinodular goiters, VEGF was detected in contact with the extracellular matrix at the basal pole of the cells. In conclusion, the present study endorses the likelihood of angiofollicular units in the human thyroids. Vascular changes are related to the functional status of thyrocytes.

Cell necrosis and apoptosis are differentially regulated during goitre development and iodine-induced involution.

Necrosis and apoptosis coexist in the thyroid during goitre development and involution, but little is known about their respective causes. To test the possible role of free radicals, we analysed separately necrosis and apoptosis in male Wistar rats with depressed or normal antioxidant protection. Vitamin E-deficient and -sufficient rats were made goitrous with perchlorate in drinking water; involution was induced by repeated injection of NaI, without or with methimazole. Increase of thyroid malondialdehyde concentration and decrease of glutathione peroxidase activity confirmed the depressed antioxidant protection in vitamin E-deficient rats. Plasma thyroxine and TSH levels were not modified. Necrosis (swollen cells) and apoptosis (pyknotic cells) were quantified on histological sections. In vitamin E-sufficient rats, dead cells were very rare in control thyroids, increased 3-fold in goitre and still further during involution. Necrotic epithelial cells predominated in the goitre and their number declined after iodide supplementation, without or with methimazole. In contrast, the number of apoptotic cells and the caspase-3 activity were increased in goitre and further increased after involution, with two-thirds of pyknotic cells being observed in the interstitium. Apoptosis was prevented by methimazole. Vitamin E deficiency significantly increased total cell death and epithelial cell necrosis and induced the occurrence of much cell debris in the follicular lumen during involution, with no modification of the apoptotic reaction. These results show that the type of cell death is differentially regulated during goitre development and involution: necrosis is related to the oxidative status of the cells, while apoptosis comes with iodine-induced involution.

Destruction of contrast microbubbles by ultrasound: effects on myocardial function, coronary perfusion pressure, and microvascular integrity.

BACKGROUND: Recent experimental data indicate that ultrasound-induced destruction of ultrasound contrast microbubbles can cause immediate rupture of the microvessels in which these microbubbles are located. METHODS AND RESULTS: To examine the functional and morphological significance of these findings in the heart, isolated rabbit hearts were perfused retrogradely with buffer containing ultrasound contrast agents and were insolated at increasing levels of acoustic energy with a broadband transducer emitting at 1.8 MHz and receiving at 3.6 MHz and operated in the triggered mode (1 Hz). At the end of each experiment, the hearts were fixed in glutaraldehyde and examined with light microscopy. Neither exposure to ultrasound alone or to contrast alone affected left ventricular developed pressure. By contrast, simultaneous exposure to contrast and ultrasound resulted in a reversible, transient mechanical index (MI)-dependent decrease in left ventricular developed pressure (to 83+/-5% of baseline at an MI of 1.6) and a transient MI-dependent increase in coronary perfusion pressure (to 120+/-6% of baseline at an MI of 1.6). Myocardial lactate release also showed significant increases with increasing MIs. Macroscopically, areas of intramural hemorrhage were identified over the beam elevation in hearts exposed to both contrast and high-MI ultrasound. Light microscopy revealed the presence of capillary ruptures, erythrocyte extravasation, and endothelial cell damage. The mean percentage of capillaries ruptured at an MI of 1.6 was 3.6+/-1.4%. CONCLUSIONS: Simultaneous exposure of isolated rabbit hearts to ultrasound and contrast agents results in an MI-dependent, transient depression of left ventricular contractile function, a rise in coronary perfusion pressure, an increase in lactate production, and limited capillary ruptures.

Neural morphological effects of long-term implantation of the self-sizing spiral cuff nerve electrode.

The paper reports on the histological effects of chronic implantation of self-sizing spiral cuff nerve electrodes on the cat sciatic nerve. The implantation period is about 4.4 months. Four different experimental conditions are evaluated: control, sham, bare cuff (cuffs without contacts and leads) and full cuff. The total number of axons in the nerves of the control group is compared with the three other groups. The surface occupied by collagen fibres in the nerve section, perineurium thickness, fibre diameter and myelin thickness are also measured. The average number of axons in the control nerves is found to be 16,416 (+/- 1,509) and does not differ significantly from the three other groups (p > 0.1). Collagen measurements show an extrafascicular epineurial fibrosis in the two implanted groups that is found to be significantly different (p < 0.05). No differences are encountered in the perineurium thickness analysis. Fibre diameter distributions show a regular bimodal pattern for all groups. Centrality (mean and Pm) and dispersion statistics (P25 and P75) extracted from fibre diameter distributions do not reveal significant differences. Myelin thickness distributions are also similar for all groups, as well as centrality and dispersion statistics. The present morphometrical results suggest that the effects produced by a chronic spiral cuff implant on this animal model are negligible.

Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle.

We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.

Evidence for co-ordinated changes between vascular endothelial growth factor and nitric oxide synthase III immunoreactivity, the functional status of the thyroid follicles, and the microvascular bed during chronic stimulation by low iodine and propylthiouracyl in old mice.

Vasoactive and angiogenic factors are involved in the autocrine/paracrine thyroid regulation of microvascular bed during goiter development. In the thyroid of old mice, the presence of slowly functioning ('cold') follicles allowed us to study the microvascular regulation of each follicle in correlation with the immunohistochemical expression of vascular endothelial growth factor (VEGF) and nitric oxide synthase III (NOSIII). Mice aged 20 months did or did not receive a goitrogenic treatment (low iodine diet and propylthiouracyl), and their thyroids were processed for light and electron microscopy, and for autoradiography. The relative volumes (Vv) of the capillaries, the number of vessels per follicular area, the mean capillary area and the number of [(3)H]thymidine labeled nuclei were measured separately for 'hot' and 'cold' follicles. Already in control mice, the capillary bed surrounding 'hot' follicles was significantly larger than that seen around 'cold' follicles, because of larger diameters and twice the number of capillaries. This difference persisted whatever the length of the stimulatory treatment. During this treatment, the Vv of the capillaries increased to a larger extent around 'hot' follicles than around 'cold' ones. All vascular changes around 'cold' follicles were less extended and the increase in the capillary diameter was delayed. In control mice, the 'cold' follicles were negative for NOSIII and positive for VEGF while 'hot' follicles were positive for both. During stimulation, all follicles became progressively NOSIII positive. These data support the concept of 'angio-follicular units' in the thyroid and demonstrate their differential regulation in chronic stimulation during which local secretion of VEGF and NO is clearly involved.

Automatic morphometry of nerve histological sections.

A method for the automatic segmentation, recognition and measurement of neuronal myelinated fibers in nerve histological sections is presented. In this method, the fiber parameters i.e. perimeter, area, position of the fiber and myelin sheath thickness are automatically computed. Obliquity of the sections may be taken into account. First, the image is thresholded to provide a coarse classification between myelin and non-myelin pixels. Next, the resulting binary image is further simplified using connected morphological operators. By applying semantic rules to the zonal graph axon candidates are identified. Those are either isolated or still connected. Then, separation of connected fibers is performed by evaluating myelin sheath thickness around each candidate area with an Euclidean distance transformation. Finally, properties of each detected fiber are computed and false positives are removed. The accuracy of the method is assessed by evaluating missed detection, false positive ratio and comparing the results to the manual procedure with sampling. In the evaluated nerve surface, a 0.9% of false positives was found, along with 6.36% of missed detections. The resulting histograms show strong correlation with those obtained by manual measure. The noise introduced by this method is significantly lower than the intrinsic sampling variability. This automatic method constitutes an original tool for morphometrical analysis.

Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves' disease.

We performed genetic immunization of outbred NMRI mice, using a cDNA encoding the human thyrotropin receptor (TSHr). All mice produced antibodies capable of recognizing the recombinant receptor expressed at the surface of stably transfected Chinese hamster ovary (CHO) cells, and sera from most of the immunized mice blocked TSH-dependent stimulation of cAMP accumulation in cells expressing the TSHr. Five out of 29 female mice showed sign of hyperthyroidism including elevated total T4 and suppressed TSH levels. The serum of these mice contained thyroid-stimulating activity, as measured in a classic assay using CHO cells expressing recombinant TSHr. In contrast, only 1 male out of 30 had moderately elevated serum total T4 with undetectable TSH values. The hyperthyroid animals had goiters with extensive lymphocytic infiltration, characteristic of a Th2 immune response. In addition, these animals displayed ocular signs reminiscent of Graves' ophthalmopathy, including edema, deposit of amorphous material, and cellular infiltration of their extraocular muscles. Our results demonstrate that genetic immunization of outbred NMRI mice with the human TSHr provides the most convincing murine model of Graves' disease available to date.

Activation of Poly(ADP-Ribose)Polymerase in rat hepatocytes does not contribute to their cell death by oxidative stress.

Oxidative stress induced by tert-butyl hydroperoxide (tBOOH) in freshly isolated rat hepatocytes caused DNA damage and loss of membrane integrity. Such DNA lesions are likely to be single strand breaks since neither caryolysis nor chromatine condensation was seen in electron micrographs from tBOOH-treated cells. In addition, pulsed field gel electrophoresis of genomic DNA from both control and tBOOH-treated hepatocytes showed similar profiles, indicating the absence of internucleosomal DNA cleavage, a classical reflection of apoptotic endonuclease activity. The activation of the repair enzyme poly(ADP-ribose)polymerase (PARP) following DNA damage by tBOOH induced a dramatic drop in both NAD(+) and ATP. The inhibition of PARP by 3-aminobenzamide enhanced DNA damage by tBOOH, restored NAD(+) and ATP levels, but did not result in better survival against cell killing by tBOOH. The lack of the protective effect of PARP inhibitor, therefore, does not implicate PARP in the mechanism of tBOOH-induced cytotoxicity. Electron micrographs also show no mitochondrial swelling in cells under oxidative stress, but such organelles were mainly located around the nucleus, a picture already observed in autoschizis, a new suggested kind of cell death which shows both apoptotic and necrotic morphological characteristics.

Protein S-thiolation can mediate the inhibition of protein synthesis induced by tert-butyl hydroperoxide in isolated rat hepatocytes.

A rapid inhibition of protein synthesis is observed when isolated rat hepatocytes are incubated in the presence of 0.25-0.5 mM of tert-butyl hydroperoxide (tBOOH). Such an inhibition occurs in the absence of a cytolytic effect by tBOOH. Iron chelators (o-phenanthroline and desferrioxiamine), protected against oxidative cell death, but they did not modify the inhibition of protein synthesis caused by tBOOH (0.5 mM), suggesting that free radicals are less implicated in such an impairment. Electron micrographs of hepatocytes under oxidative stress show disaggregation of polyribosomes but not oxidative alterations, such as blebs or mitochondrial swelling. Protein synthesis inhibition is accompanied by a decrease in reduced glutathione (GSH) and an increase in glutathione disulfide (GSSG) and the level of protein S-thiolation (protein mixed disulfides formation). Such an increase of GSSG appears as a critical event since diethylmaleate (DEM) at 0.2 mM reduced GSH content by more than 50% but did not affect either GSSG content or protein synthesis. The addition of exogenous GSH and N-acetylcysteine (NAC) to tBOOH-treated hepatocytes significantly reduced the formation of protein mixed disulfides and restored the depressed protein synthesis either completely or partially. We suggest that S-thiolation of some key proteins may be involved in protein synthesis inhibition by tBOOH.

Detection of Mycobacterium avium subsp. paratuberculosis in infected tissues by new species-specific immunohistological procedures.

We have previously described the cloning and sequencing of a gene portion coding for the terminal part of a 34-kDa protein of Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne's disease (P. Gilot, M. De Kesel, L. Machtelinckx, M. Coene, and C. Cocito, J. Bacteriol. 175:4930-4935, 1993). The recombinant polypeptide (a362) carries species-specific B-cell epitopes which do not cross-react with other mycobacterial pathogens (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol. 31:947-954, 1993). The present work describes the preparation of polyclonal and monoclonal antibodies directed against a362 and the use of these immunoglobulins for histopathological diagnosis of Johne's disease. The new immunohistological procedures herewith detailed proved to be able to identify M. avium subsp. paratuberculosis antigens in the intestinal tissues and lymph nodes of cattle affected by either the paucibacillary or pluribacillary form of the disease. They yielded negative responses not only with healthy animals but also with those affected by tuberculosis (Mycobacterium bovis). Both immunohistological procedures proved to be as sensitive as or more sensitive than Ziehl-Neelsen staining and, in addition, to be endowed with species specificity.

Antigoitrogenic effect of combined supplementation with dl-alpha-tocopherol, ascorbic acid and beta-carotene and of dl-alpha-tocopherol alone in the rat.

The effects of the vitamins dl-alpha-tocopherol, ascorbic acid and beta-carotene, free radical scavengers and lipid peroxidation inhibitors, were analyzed in male Wistar rats made goitrous by feeding a low iodine diet (< 20 micrograms iodine/kg) and perchlorate (1% in drinking water) for 4, 8, 16, and 32 days. Groups of control or goitrous rats received for at least 16 days before killing a diet containing 0.6% vitamin E (as dl-alpha-tocopherol acetate), 1.2% vitamin C (ascorbic acid) and 0.48% beta-carotene, either simultaneously (vitamin cocktail) or separately. This treatment led to a 5-fold increase of vitamin E in the thyroid gland, a 24-fold increase in the liver and a 3-fold increase in the plasma. In control rats, vitamin cocktail administration increased slightly the thyroid weight with little changes in thyroid function parameters. During iodine deficiency, administration of the vitamin cocktail or vitamin E alone reduced significantly the rate of increase in thyroid weight, and DNA and protein contents, as well as the proportion of [3H]thymidine labeled thyroid follicular cells, but not that of labeled endothelial cells. Plasma tri-iodothyronine, thyroxine, TSH levels, thyroid iodine content and concentration as well as relative volumes of glandular compartments were not modified. The proportion of necrotic cells rose from 0.5% in normal animals to about 2% after 16 days of goiter development. No significant protective effect of the vitamins was observed. These results suggest that these vitamins, particularly vitamin E, modulate one of the regulatory cascades involved in the control of thyroid follicular cell growth, without interfering with the proliferation of endothelial cells.

Costimulation of adenylyl cyclase and phospholipase C by a mutant alpha 1B-adrenergic receptor transgene promotes malignant transformation of thyroid follicular cells.

Proliferation of thyroid follicular cells is controlled by three intra-cellular cascades [cAMP, inositol 1,4,5-triphosphate (IP3)/Ca2+/diacylglycerol (DAG), and tyrosine kinases] that are activated by distinct extracellular signals and receptors. We had previously generated a transgenic mouse model in which the cAMP cascade was permanently stimulated in thyroid cells by an adenosine A2a receptor (Tg-A2aR model). In the present work, we have generated a transgenic model characterized by the chronic stimulation of both adenylyl cyclase and phospholipase C in thyroid follicular cells. The bovine thyroglobulin gene promoter was used to direct the expression of a constitutively active mutant of the alpha 1B adrenergic receptor, which is known to couple to both cascades in transfected cell lines. The expression of the transgene resulted, as expected, in the activation of phospholipase C and adenylyl cyclase, as demonstrated by the direct measurement of IP3 and cAMP in thyroid tissue. The phenotype resulting from this dual stimulation included growth stimulation, hyperfunction, cell degeneracy attributed to the overproduction of free radicals, and the development of malignant nodules invading the capsule, muscles, and blood vessels. Differentiated metastases were found occasionally in old animals. The development of malignant lesions was more frequent and of earlier onset than in our previous Tg-A2aR model, in which only the cAMP cascade was stimulated. These observations demonstrate that the cAMP and IP3/Ca2+/DAG cascades can cooperate in vivo toward the development of thyroid follicular cell malignancies.

Cyclic feeding behaviour and changes in hypothalamic galanin and neuropeptide Y gene expression induced by zinc deficiency in the rat.

Dietary zinc-deficiency induces a striking reduction and a cyclic pattern of food intake in rodents. To elucidate the mechanisms for these effects, we studied the hypothalamic content, synthesis, and distribution of galanin (GAL) and neuropeptide Y (NPY) during zinc deficiency and refeeding in the rat. In Wistar rats, three weeks of zinc-deprivation consistently induced a reduction and a cyclic pattern of night- and day-time food intake, as well as of water intake. This was accompanied in zinc-deficient (ZD) rats, and to a lesser extent in pair-fed (PF) rats, by a decrease of hypothalamic GAL mRNA concentration (CTR: 100 +/- 8, ZD: 61 +/- 4, PF: 78 +/- 2 arbitrary densitometric units, ADU, P < 0.01) and an increase of hypothalamic NPY (CTR: 100 +/- 11, ZD: 154 +/- 10, PF: 126 +/- 4 ADU, P < 0.05), without peptide modification. The two neuropeptidergic systems were not affected by the cycles of feeding, with the exception of the NPY-immunoreactivity in the suprachiasmatic nuclei (geniculo-hypothalamic tract), that was inversely correlated to the food intake in both ZD and PF animals. In a second experiment, we showed that zinc-repletion for 4 days suppressed the behaviour induced by a two-week zinc-deprivation, and reversed the increase of NPY mRNA in ZD animals. We finally demonstrated that zinc-deficiency induced a similar behaviour in Zucker rats. However, in these rats whose synthesis of NPY is constitutively up-regulated, no change of NPY synthesis was observed in ZD rats, suggesting that the increase observed in Wistar is adaptative rather than instrumental to the abnormal food intake. In conclusion, we have further characterized the cyclic feeding behaviour of the zinc-deficient Wistar rats, and shown in these animals a decreased activity of the GAL system and an increased activity of the NPY system, likely corresponding to a compensatory response of the two neuropeptidergic systems, as observed in food-deprived animals. As spontaneous food intake of ZD rats does not increase, a resistance to NPY could also be present. These behavioural and neuropeptidergic changes were partially reversed by reintroduction of zinc in the diet. In Zucker rats, the same behaviour occurred despite an insensitivity of the NPY system to the zinc-deficiency. In addition, we describe a nutritional regulation of the NPY-immunoreactivity in the geniculo-hypothalamic tract, that could constitute the substrate of circadian rhythm modulation by timed feeding.

Selenium deficiency and thyroid fibrosis. A key role for macrophages and transforming growth factor beta (TGF-beta).

Free radical damage and fibrosis caused by selenium deficiency are thought to be involved in the pathogenesis of myxoedematous cretinism. So far, no pathway explains the link between selenium deficiency and tissue fibrosis. Pharmacological doses of iodine induce necrosis in iodine-deficient thyroids. Necrosis is much increased if the glands are also selenium-deficient, which then evolve to fibrosis. This rat model was reproduced to explore the role of selenium deficiency in defective tissue repair. At first, proliferation indexes of epithelial cells and fibroblasts were comparable between selenium-deficient and control groups. Then, in selenium-deficient thyroids the inflammatory reaction was more marked being mainly composed of macrophages. The proliferation index of the epithelial cells decreased, while that of the fibroblasts increased. These thyroids evolved to fibrosis. TGF-beta immunostaining was prominent in the macrophages of selenium-deficient rats. Anti TGF-beta antibodies restored the proliferation indexes, and blocked the evolution to fibrosis. In selenium deficiency, an active fibrotic process occurs in the thyroid, in which the inflammatory reaction and an excess of TGF-beta play a key role.

Early occurrence of metastatic differentiated thyroid carcinomas in transgenic mice expressing the A2a adenosine receptor gene and the human papillomavirus type 16 E7 oncogene.

We report here the characterization of a transgenic mouse model (Tg-A2aR/Tg-E7) resulting from the coexpression of two oncogenic transgenes in the thyroid. The two transgenes (Tg-A2aR and Tg-E7) were placed under control of the thyroid specific thyroglobulin gene promoter, and directed the expression of either the A2a adenosine receptor that constitutively activates the cAMP pathway, or the E7 protein of the human papillomavirus type 16, that binds and inactivates the retinoblastoma susceptibility gene product (Rb1). Transgenic mice expressing both transgenes were generated by interbreeding the Tg-A2aR and Tg-E7 transgenic lines, generated and characterized previously (Ledent et al., 1992, 1995). These mice develop a larger goiter than that of the two parental lines, and a severe hyperthyroidism comparable to that observed in the Tg-A2aR parental line. The main feature of the Tg-A2aR/Tg-E7 mice is the rapid occurrence of malignant lesions, and the dissemination of malignant thyroid tissue through the blood stream, generating multiple differentiated and functional metastases in the lung. These metastases appeared as early as 2 months after birth and their frequency increased to 75% over 3 months. They were associated with the presence of large vascular lakes in the thyroid. Electron microscopy of the malignant cells revealed nuclear features similar to those of human thyroid papillary carcinoma. These mice, in which two oncogenes are co-expressed in the thyroid, represent the first genetic animal model developing metastatic differentiated carcinomas of the thyroid with a high frequency.