SUMMARY: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 ␣ (TNF␣) 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. (Lab Invest 2000, 80:1215-1225.

Serum thyroglobulin (Tg) concentration increases in diverse thyroid pathophysiological situations. We examined whether Tg molecules appearing in the serum of patients with Graves' disease (GD), subacute thyroiditis (ST) or differentiated thyroid cancer (DTC) have distinctive biochemical properties. We used ultracentrifugation on sucrose gradient and Western blot to analyze structural parameters of immunoreactive Tg in complete serum from 40 patients with GD, ST or DTC. Purified human Tg was used as reference.Immunoreactive Tg from ST or DTC sera sedimented in a single, rather symmetrical peak as purified 19S Tg. In GD sera without detectable anti-Tg autoantibodies (TgaAb), about 80% of immunoreactive Tg was recovered in a Tg dimer peak that often split into two components; the remaining Tg immunoreactivity (10-30%) sedimented faster and was polydispersed. In GD sera containing TgaAb, immunoreactive Tg was recovered in a peak sedimenting faster than 19S Tg corresponding to immune complexes identified by protein A adsorption. Using a Western blot procedure, optimized to detect 0.1 ng Tg in serum, a single band of Tg, migrating as the intact Tg subunit, was always found in nonreducing conditions; the intensity of the band correlated with the immunoassayable Tg concentration. In reducing conditions, the Tg band obtained with GD or ST sera was decreased by up to 70% compared with that of purified Tg or serum Tg from patients with DTC.In conclusion, serum Tg from DTC is remarkably homogeneous and in the form of dimers dissociable into uncleaved monomers. In contrast, serum Tg from GD or ST is heterogeneous with respect to its sedimentation properties and/or the structural integrity of its polypeptide chains. These data provide information on the processes whereby Tg is released into the circulation.

Nitric oxide (NO) is a well-known mediator of autoimmune processes. In the thyroid gland, it is produced in response to interleukin 1 (IL-1) and may mediate cytokine action at an early stage of autoimmune thyroiditis. In this study, we have investigated whether NO is involved in cytokine-induced cytotoxic effects and epithelial barrier alterations in thyrocytes. Human thyroid epithelial cells were cultured as tight polarised monolayers on a permeable support and exposed or not to IL-1 (100 U/ml), alone or in combination with interferon-(IFN-; 100 U/ml) added to the basal compartment. NO production was not detected in control thyrocytes, but was significantly induced by the combination of IL-1 with IFN-, in a time-dependent fashion. Similarly, expression of the inducible isoform of nitric oxide synthase (NOSII), determined by immunoblot and immunofluorescence confocal microscopy, was not detected in control cells, but was markedly induced after 48-h exposure to both cytokines. This treatment significantly increased the release of cytosolic lactate dehydrogenase (LDH) in the apical and basolateral media and decreased transepithelial electrical resistance. Although IFN-was not sufficient to induce NO production, it could by itself decrease transepithelial resistance and synergised the IL-1 effect on LDH release. The NOS inhibitor, -nitro-argininemethyl ester, suppressed the cytokine-induced NO production and decreased the LDH release, but failed to prevent the loss of transepithelial resistance. These results indicated that human thyrocytes express NOSII and produce NO in response to IL-1 +IFN-and suggest that NO acts as a mediator of cytokine-induced cytotoxicity in the thyroid gland and may promote the exposure of autoantigens to the immune system. In contrast, NO does not appear to mediate the cytokine-induced disruption of the thyroid epithelial barrier.

Although many studies have investigated the metabolism and bioavailability of Si supplemented in foods or as a food ingredient, this was the first to investigate and demonstrate the digestibility of OSA administered in a complex form with vanillin.

SUMMARY: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-␤, 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. (Lab Invest 2001, 81:1601-1613.