Bernadette Nabarra

Persistence of Ca2+-Sensing Receptor Expression in Functionally Active, Long-Term Human Parathyroid Cell Cultures

An original human parathyroid cell culture model from uremic patients with II° hyperparathyroidism has been developed, with its main feature being long‐term functionally active viability up to 5 months, as assessed by persistent responsiveness to changes of extracellular Ca2+ concentrations ([Ca2+]e). In addition to the inhibitory effect of increasing [Ca2+]e, increasing extracellular phosphate exerted a biphasic effect on parathyroid hormone (PTH) secretion. The presence of the Ca2+‐sensing receptor (CaR), on which depends the response to [Ca2+]e and its persistence, has been demonstrated in our culture system both by direct detection and by inhibition of its activity. CaR protein was detected by Western blot analysis with a specific anti‐CaR antibody. CaR gene transcripts have been identified by reverse transcription‐polymerase chain reaction analysis. mRNA (by in situ hybridization) and protein (by immunocytochemistry) expression were detected for both CaR and PTH. Adding a specific anti‐CaR antibody to the medium induced a marked reduction of low [Ca2+]e‐stimulated PTH release, which decreased to levels equivalent to those obtained in high [Ca2+]e medium. The described long‐term functionality could be due to several factors, including the clustered cell type of culture yielded by our preparation procedure, the growth characteristics of hyperplastic uremic tissue, and the use of a phosphate‐rich medium. The present model, because of its long‐term functionality, is a unique tool for the exploration of PTH synthesis and secretion and for studies of parathyroid cell growth in vitro.

Pattern of secretion in thymic epithelial cells: Ultrastructural studies of the effect of blockage at various levels

SummaryThe observation of secretory phenomena in mouse thymic epithelial cells is disappointing since no real secretion image is found. An adequate technique for such a study is to block the secretion pathway and to observe by electron microscopy cells accumulating secretory products. For this purpose, we used three means of blocking secretion: Firstly, since the thymic epithelial cell is regulated by a feedback phenomenon, secretion was blocked by antibodies against thymulin, one of the hormones secreted by these cells. Secondly, colchicine was used to modify the intracellular transport of the secretory product. In both of these types of experiments, electron microscopy showed a great increase in the number of “clear vacuoles” and their granular contents in epithelial cells. In a third series of experiments, we used monensin at a concentration that blocks the intracellular transport of secretory proteins at the various levels of the Golgi apparatus. In this series, only an increased number of vacuoles was observed, but they appeared devoid of all granular content. It can be concluded that in the thymic epithelial cell, a discrete system of secretion directs the passage of the product, originating in the cisternae of the endoplasmic reticulum, into “clear vacuoles”, the terminal element of the cellular secretory apparatus.

Localization of zinc in the thymic reticulum of mice by electron-probe microanalysis

SummaryThymulin, a thymic hormone, is a nonapeptide requiring zinc for biological activity. It has been shown that epithelial cells, forming part of the thymic reticulum, secrete this hormone and/or store it within cytoplasmic vacuoles. X-ray electron-probe microanalysis (EPMA) has been used to detect zinc in the thymus. Low concentrations of zinc have been demonstrated in the dense granules contained in clear vacuoles of some epithelial cells in normal and ZnCl2-injected mouse thymuses, thus suggesting that the metal may be coupled to the peptide before the secretion of the hormone from the cells.

Effect of cytokines on thymic hematopoietic precursors

SummaryWe have previously shown that the interaction of thymocytes with thymic accessory cells (macrophages and/or interdigitating cells) is one of the factors required for thymocyte activation. Precursors of both thymic accessory cell and thymocytes are included in the CD4- CD8- Mac-1- Ia- subpopulation, and their respective maturation and/or activation may be modulated by granulocyte-macrophage colony-stimulating factor, interleukin 1 and interleukin 2. When CD4- CD8- thymic cells are activated with granulocyte-macrophage colony-stimulating factor plus interleukin 2, both macrophages and interdigitating-like cells are present, as shown by electron microscopy. When activated with interleukin 1 plus interleukin 2, the interdigitating-like cells is the only accessory cell present. In both culture conditions, large clusters are formed between interdigitating cells and lymphoid cells. These results have led us to propose two-step signals for thymocyte proliferation: first, the maturation of macrophages under granulocyte-macrophage colony-stimulating factor control and the production of interleukin 1, and secondly, the maturation of interdigitating cells under interleukin 1 control, their clustering with thymocytes which are then activated.

Ultrastructural study on long-term cultures of bone marrow cells with histamineproducing stimulating factor (HCSF)

SummaryIn a previous study (Dy et al. 1981) we have demonstrated that histamine-producing cell stimulating factor (HCSF), a lymphokine released by T-cells, is present in supernatants obtained from secondary mixed lymphocyte cultures set up with cells from the donor and the recipient of a skin allograft, and that HCSF causes an increased production of histamine from target cells present in bone marrow. The most abundant source of target cells was found in the less dense layer of a discontinuous Ficoll gradient of bone marrow cells. Ultrastructural studies of this layer showed that it is composed of four types of cell : type I, immature cells; type II, mastocyte-like cells; type III, macrophages and type IV, lymphocytes. We have examined the effect of HCSF on this cell population; in long-term cultures we observed a progressive numerical decrease in type I cells, accompanied by an increase in type II cells (clearly observed as early as 48 h), leading to a pure population of mastocytes cells after 45 days of culture.

Morphological and Functional Impairment of the Developing Rat Kidney Exposed to Gentamicin in Utero

Aminoglycoside antibiotics used to treat severe bacterial infection during pregnancy can cross the placenta. The effects of in utero exposure to these compounds is receiving increasing attention. It was shown that in the foetal animal, as in the adult, the kidney was the major site of gentamicin accumulation (1). In foetal guinea-pigs whose mothers were given daily 4 mg/kg of gentamicin for the seven days immediately following the period of nephrogenesis, intrarenal gentamicin concentration was found to be about 2 ug/g of tissue. More than two weeks after the treatment to the mother has ceased, the antibiotic released from all maternal and foetal tissues continued to accumulate in the developing kidney, especially as the renal blood flow and glomerular filtration rate increased with age (2). In pups born of gentamicin-treated mothers, impairments of the growth and the function of the proximal tubules were observed, but the damage was transitory (2).

The Dense Deposits Disease of the Renal Basement Membranes

Dense-deposits disease is characterized by an original morphological aspect of the renal basement membranes which have an electron-dense appearance. The immunofluorescence studies showed C3 alone in kidney. Low serum C3 levels and the presence of C3 NF activity have been detected in this disease, demonstrating an activation of the complement alternative pathway. The observation of recurrence in transplanted kidneys contributed to establish the sequence of the morphological events and showed the dissociation between the serological complement profiles and the lesions. Although the nature of the dense deposits remains still unknown, the altered membranes are recognized by at least some anti-GBM antibodies.