ABSTRACT

The effects of TSH and forskolin were examined on intracellular free calcium ([Ca²⁺]_i) and calmodulin in normal pig thyroid cells in culture. TSH was found to produce acute increases in [Ca²⁺]_i in pig cells. Responses were seen at concentrations of TSH between 0·01 and 10 mU/ml. Sensitivity to TSH was greater in adherent monolayers of cells than in cell suspensions and was also greater in subconfluent rather than confluent monolayers of cells. The increase in [Ca²⁺]_i in response to TSH represented just over a doubling in [Ca²⁺]_i whether examined at 22 °C or 37 °C. Forskolin failed to affect [Ca²⁺]_i. TSH increased [Ca²⁺]_i in the absence of extracellular calcium.

TSH, but not forskolin, produced a significant increase in intracellular calmodulin after 3 days of culture of cells with TSH. The increase in calmodulin was of the order of 60% and did not relate to any effect of TSH on thyroid cell number.

Journal of Endocrinology (1991) 129, 291–299

During pregnancy, immune activity is tightly regulated so that antimicrobial protection of the mother and fetus is balanced with the need for immune tolerance to prevent fetal rejection. In this setting, the maternal–fetal interface, in the form of the uterine decidua, provides a heterogeneous immune cell population with the potential to mediate diverse activities throughout pregnancy. Recent studies have suggested that vitamin D may be a key regulator of immune function during pregnancy, with the fetal–maternal interface representing a prominent target. Among its non-classical actions are potent immunomodulatory effects, including induction of antibacterial responses and modulation of T-lymphocytes to suppress inflammation and promote tolerogenesis. Thus, vitamin D may play a pivotal role in normal decidual immune function by promoting innate responses to infection, while simultaneously preventing an over-elaboration of inflammatory adaptive immunity. Research to date has focused upon the potential role of vitamin D in preventing infectious diseases such as tuberculosis, as well as possibly suppressing of autoimmune disease. Nevertheless, vitamin D may also influence facets of immune function not immediately associated with primary innate responses. This review summarises our current understanding of decidual immune function with respect to the vitamin D metabolism and signalling, and as to how this may be affected by variations in maternal vitamin D status. There has recently been much interest in vitamin D supplementation of pregnant women, but our knowledge of how this may influence the function of decidua remains limited. Further insight into the immunomodulatory actions of vitamin D during pregnancy will help shed light upon this.

We report on the culture of human insulinoma cells derived from a 32-year-old male patient with hyperinsulinism due to an insulinoma of the pancreas. A single-cell suspension was made by passing insulinoma fragments through a fine-gauge stainless-steel mesh. Cluster-forming insulinoma cells resembling pancreatic islets grew in the presence of fibroblasts. The insulinoma cell clusters could be differentiated from fibroblasts by using in situ pan optic staining and specific immunocytochemical staining (anti-human insulin and anti-human insulinoma monoclonal antibody (mAb) D24). mAb D24 was generated using insulinoma cells as antigen for immunization of a Balb/C mouse and cell fusion by the hybridoma cell technique. The anti-insulinoma cell mAb recognized a 32 kDa protein on immunoblot analysis of neuroendocrine tumor cells. D24 mAb also reacted immunohistochemically with normal pancreatic beta-cells and tumors such as vipoma, gastrinoma and carcinoid. Insulinoma cell clusters separated from fibroblasts by micromanipulation and plated into multiwell culture dishes exhibited an insulin-secretion rate of approximately 30 U/100 cells per 24 h with no insulin-secretory response to elevated glucose concentration. Purified insulinoma cells incubated with 1 ng/ml human nerve growth factor expressed neurofilament and neurite extension. These findings together with earlier observations in animal models suggest that human pancreatic beta-cells share some properties with neurons and are related to other neuroendocrine cells in the gastrointestinal tract.

Abstract

Hyperthyroidism is associated with elevated plasma levels of endothelium-derived proteins such as von Willebrand factor (vWF), fibronectin (FN) and endothelin-1 (ET-1). This study was designed to characterize the mechanisms involved in this phenomenon at the cellular level. vWF, FN and ET-1 secretion and mRNA expression were measured in human umbilical vein endothelial cells (HUVECs) exposed to tri-iodothyronine (T₃) for 13 ± 1 days, using ELISA, Western blot, RIA and Northern blot analysis respectively. Exposure of HUVECs to T₃ significantly increased vWF secretion (50 ng T₃/ml: 117 ± 5%, P<0·01; 100 ng T₃/ml: 127 ± 26%, P<0·01) as well as vWF mRNA expression (50 ng/ml: 116 ± 13%, P<0·001; 100 ng/ml: 136 ± 30%, P<0·002) (results are means ± s.d. analysed by the Wilcoxon signed rank test). FN secretion was significantly affected by 50 (145 ± 42% of control, P<0·05) and 100 (116·8 ± 16% of control, P<0·05) ng T₃/ml, and FN mRNA expression by 50 ng T₃/ml (123 ± 20%, P<0·05). Long-term incubation with T₃ increased both ET-1 secretion (25 ng/ml: 124 ± 25%, P<0·001; 50 ng/ml: 165 ± 53%, P<0·05; 100 ng/ml: 116 ± 17%, P<0·05) and prepro-ET-1 mRNA expression (25 ng/ml: 112 ± 16%, P<0·05; 50 ng/ml: 134 ± 43%, P<0·02; 100 ng/ml: 120 ± 20%, P<0·02). Protein kinase C (PKC) isoforms ε and βII were not significantly affected by T₃, whereas PKC α was increased in whole cell lysates and in membrane fractions of cells incubated with 100 but not 50 ng T₃/ml. Prepro-ET-1 mRNA stability, cell numbers and proliferation, measured by [³H]thymidine assays, remained unaffected in HUVECs after exposure to T₃. These data indicate thyroid hormone-induced upregulation of mRNA expression and protein synthesis of vWF, FN and ET-1, by PKC α-, βII- and ε-independent pathways, explaining, at least in part, increased plasma concentrations of endothelial proteins and peptides in the hyperthyroid state.

Journal of Endocrinology (1997) 154, 231–239

Abstract

While the multifunctional proteins of the transforming growth factor-β (TGF-β) family have a potent antiproliferative effect on thyroid follicular cell growth, increased expression of TGF-β in proliferating thyroid cells and in thyroid tumours has recently been described, suggesting a secondary counter-regulatory role of these proteins. We have studied further this apparent paradox in vitro using FRTL-5 cells, 5 continuous cell strains from feline multinodular goitres (MNG) and 29 primary cultures prepared from human MNG.

While dose dependent inhibition of FRTL-5 cell growth was confirmed, a variable fraction of these cells was naturally resistant towards TGF-β1, thus explaining the large interassay variability of growth inhibition (36 to 98% within 2 days, n=19). After 40 days of continuous exposure, FRTL-5 cells became fully refractory towards TGF-β1 inhibition, due to the selective growth of naturally resistant subclones, as demonstrated for example by microscopic observation of three-dimensionally growing collagen-embedded cell clusters. The refractoriness could still be demonstrated even after several cell passages. In addition, 2 out of 5 feline thyroid cell strains obtained from feline MNG and 18 out of 29 primary cultures from human MNG showed a high degree of refractoriness towards TGF-β.

We conclude that constitutively TGF-β resistant cells may occur in thyroid glands and that persistent TGF-β refractoriness may secondarily be acquired. Resistant cells may escape regular growth control mechanisms and hence may contribute to the notorious heterogeneity of thyroid growth and to nodular transformation.

Journal of Endocrinology (1996) 149, 485–496

Experimental testing of growth, metastatic progression and drug responsiveness of human breast cancer in vivo is performed in immunodeficient mice. Drug candidates need to show promise against human breast cancer in mice before being allowed into clinical trials. Breast cancer growth is under endocrine control by ovarian steroids and the pituitary peptide hormone prolactin. While it is recognized that the most relevant biologic effects of prolactin are achieved with prolactin from the matching species, the biologic efficacy of mouse prolactin for human prolactin receptors has not been recorded. Thus, it is unclear whether the mouse endocrine environment adequately reflects the hormonal environment in breast cancer patients with regard to prolactin. We now show both recombinant and natural pituitary-derived mouse prolactin to be a poor agonist for human prolactin receptors. Mouse prolactin failed to induce human prolactin receptor-mediated biologic responses of cell clustering, proliferation, gene induction and signal transduction, including activation of Stat5, Stat3, Erk1/2 and Akt pathways. Consistent data were derived from human breast cancer lines T-47D, MCF-7 and ZR-75.1, as well as human prolactin receptor-transfected COS-7 and 32D cells. Failure of mouse prolactin to activate human prolactin receptors uncovers a key deficiency of the mouse endocrine environment for human xenotransplant studies. Since most human breast cancers express prolactin receptors, human breast cancer transferred into mice is unnaturally selected for growth in the absence of circulating prolactin. The new insight raises concerns about the validity of analyzing biology and drug responsiveness of human breast cancer in existing mouse xenotransplant models.