Iron absorption by CaCo 2 cells cultivated in serum-free medium as in vitro model of the human intestinal epithelial barrier.

A cell culture system consisting of confluent monolayer of human enterocyte-like CaCo 2 cells, cultivated in a serum-free nutritive medium, on microporous synthetic membranes has been used as an in vitro model of the intestinal epithelial barrier. The uptake of 55ferric citrate, as well as the transepithelial passage from the apical to the basolateral pole, have been studied. CaCo 2 cells accumulate iron in a time- and concentration-dependent process, largely specific from the apical pole.

Phase I and II biotransformations in living CaCo 2 cells cultivated under serum-free conditions. Selective apical excretion of reaction products.

CaCo 2 cells, cultivated in a synthetic, serum-free nutritive medium on poly (ethylene terephthalate) membranes, form a confluent monolayer of differentiated cells, with the apical and basolateral poles exposed to the upper and lower compartments, respectively, of bicameral culture inserts (Halleux and Schneider, In Vitro Cell Dev Biol, 27A: 293-302, 1991). This cell culture system allows the passage of intact mannitol by the paracellular route and the transcellular diffusion of testosterone which appears mainly as a biotransformed unconjugated metabolite. When ethoxyresorufin is added to either the apical or basolateral poles of living CaCo 2 cells, resorufin is formed, and more than 80% is excreted at the apical pole.

Mucormycosis during deferoxamine therapy is a siderophore-mediated infection. In vitro and in vivo animal studies.

This study investigates the pathophysiology of mucormycosis caused by Rhizopus, which has been reported in 46 dialysis patients, while treated with deferoxamine (DFO). This drug aggravates mucormycosis, which we experimentally induced in guinea pigs and which lead to a shortened animal survival (P < or = 0.01).

Estrogen and insulin modulation of intracellular insulin-like growth factor binding proteins in human breast cancer cells: possible involvement in lysosomal hydrolases oversecretion.

Differences in insulin-like growth factor binding proteins (IGFBPs) expressed within estrogen receptor positive (ER+, MCF-7/6) and negative (ER-, MDA-MB-231) human breast cancer cells cultured in chemically defined medium were observed. In the absence of insulin, 17 beta-estradiol affects this expression in ER+ cells by significantly reducing 34 and 28 kDa species. In ER+ cells, insulin appears to minimize the estrogen induced reduction of these 34 and 28 kDa IGFBPs and stimulates a 24 kDa type.

Iron absorption by intestinal epithelial cells: 1. CaCo2 cells cultivated in serum-free medium, on polyethyleneterephthalate microporous membranes, as an in vitro model.

Iron absorption by intestinal epithelial cells, passage onto plasmatic apotransferrin, and regulation of the process remain largely misunderstood. To investigate this problem, we have set up an in vitro model, consisting in CaCo2 cells (a human colon adenocarcinoma line, which upon cultivation displays numerous differentiation criteria of small intestine epithelial cells). Cells are cultivated in a serum-free medium, containing 1 microgram/ml insulin, 1 ng/ml epidermal growth factor, 10 micrograms/ml albumin-linoleic acid, 100 nM hydrocortisone, and 2 nM T3 on new, transparent, Cyclopore polyethyleneterephthalate microporous membranes coated with type I collagen.

Monoclonal antibody production in semi-continuous serum- and protein-free culture. Effect of glutamine concentration and culture conditions on cell growth and antibody secretion.

We have recently shown that the semi-continuous cultivation of a mouse hybridoma line in spinner flasks, with a basal defined medium (BDM) devoid of serum and protein, increases the secretion of the immunoreactive monoclonal antibody (MAb) by a factor of ca. 2.4, compared to culture in flasks with serum-containing medium (Schneider, 1989).

Desferrithiocin and desferrioxamine B. Cellular pharmacology and storage iron mobilization.

3H-Desferrithiocin (DFT) has been synthesized from desmethyl desferrithiocin. The uptake and release of this 3H siderophore and of its iron complex have been studied in cultured rat hepatocytes and systematically compared to 14C desferrioxamine B (DFO). At 37 degrees, the uptake of both chelators is strictly proportional to the extracellular concentration and no toxicity is observed up to, at least, 1 mM.

Antibiotic susceptibility of Salmonella spp. at different pH values.

We have examined the effects of acidic pH, in the range of those prevailing within phagosomes and lysosomes, on the growth and the susceptibility to different antibiotics of several strains of Salmonella spp. The minimal inhibitory concentration and the minimal bactericidal concentration of several beta-lactams were increased considerably during culture at pH 5.2.

Cytotoxic activity of daunorubicin or vindesin conjugated to a monoclonal antibody on cultured MCF-7 breast carcinoma cells.

Conjugates were constructed between daunorubicin or vindesin and a monoclonal antibody to human milk fat globule membrane associated antigen. This antibody recognizes a high molecular weight glycoprotein present at the cell surface of human normal and tumour epithelial cells; after specific binding to plasma membrane of cultured MCF-7 human breast carcinoma cells, it is endocytosed and gains access to lysosomes, wherein it is broken down (Aboud-Pirak et al., Cancer Res 48: 3188-3196, 1988).

Optimisation of hybridoma cell growth and monoclonal antibody secretion in a chemically defined, serum- and protein-free culture medium.

Monoclonal antibodies (MAbs), for human use require chemical and biological purity. The best approach seems in vitro cultivation in a serum-, protein-free medium. A basal defined culture medium has been developed to sustain optimal hybridoma cell growth and MAb secretion.

Binding and endocytosis of a monoclonal antibody to a high molecular weight human milk fat globule membrane-associated antigen by cultured MCF-7 breast carcinoma cells.

The aim of this study was to analyze whether a monoclonal antibody to human milk fat globule membrane-associated antigens, recognized specifically and homogeneously by human breast carcinoma cells but also by normal epithelial cells active in secretion, could be used to restrict the access of antitumoral drugs to cells exposing the epitope. The drug-antibody conjugate to be used is constructed by means of a covalent peptidic linkage stable in extracellular medium but hydrolyzed by lysomal enzymes after endocytosis of the drug-carrier conjugate. This monoclonal antibody specifically immunoprecipitates radioactive material from MCF-7 cells biosynthetically radiolabeled with galactose, glucosamine, palmitic acid, or acetic acid but not with mannose, leucine, or methionine.

Uptake, subcellular distribution and biotransformation of 3H-labelled astemizole in cultured rat hepatocytes.

When incubated with 3H-astemizole, a potent antagonist of H1 receptor, cultured rat hepatocytes, which do not express specific receptors for this ligand, avidly take up 3H-label proportionally to the drug concentration. HPLC analysis indicates that at 10 ng 3H-astemizole/ml, cells almost entirely deplete the culture medium of the drug within 4 hr of incubation. At 37 degrees, astemizole is metabolized and released into the culture medium mainly under the form of glucuronoconjugated metabolites.

Subcellular localization of transferrin protein and iron in the perfused rat liver. Effect of Triton WR 1339, digitonin and temperature.

The subcellular localization of 3H-labelled 59Fe-loaded transferrin accumulated by the liver has been studied by means of cell fractionation techniques. More than 96% of the 59Fe present in the liver of rats perfused with 59Fe-labelled transferrin is recovered in the parenchymal cells. Rat livers were perfused with 10 micrograms/ml 3H-labelled 59Fe-saturated transferrin, homogenized separated in nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and supernatant (S) fractions; M, L and P fractions were further analysed by isopycnic centrifugation in sucrose gradients.

Cellular pharmacology of deferrioxamine B and derivatives in cultured rat hepatocytes in relation to iron mobilization.

Two radiolabelled derivatives of deferrioxamine B (DF) have been synthesized: methyl-DF and acetyl-DF. Both derivatives are non cytotoxic and stable in cell culture but they are degraded in human plasma and more extensively in rat plasma. Methyl-DF, acetyl-DF and DF mobilize radioiron to the same extent from hepatocytes loaded with 59Fe citrate in the same range of extracellular concentrations.

Receptor-mediated endocytosis of polymeric IgA and galactosylated serum albumin in rat liver. Evidence for intracellular ligand sorting and identification of distinct endosomal compartments.

Rat polymeric IgA (pIgA) and galactosylated bovine serum albumin (GalBSA), once injected to rats, are avidly taken up by hepatocytes via receptor-mediated endocytosis. Of injected pIgA, 64% was transferred undigested into bile within 3 h, with a peak at 30-45 min. GalBSA was essentially digested in lysosomes.

Iron mobilization from cultured hepatocytes: effect of desferrioxamine B.

When cultured rat hepatocytes prelabelled for different times at 37 degrees with 59Fe are reincubated for 1 hr in a fresh medium, radiolabelled iron is released in the washout medium as a function of the prelabelling time, and behaves like low molecular weight material on isokinetic centrifugation in sucrose gradients. When apotransferrin or desferrioxamine B are present in the reincubation medium, the kinetics of iron release are similar but the absolute amounts of radiolabelled iron found in the culture medium are much greater. In the presence of apotransferrin, most of the 59Fe released from the cells distributes as transferrin whereas with desferrioxamine B, almost all the 59Fe is extracted by benzyl alcohol indicating its chelation by the drug.

Transferrin protein and iron uptake by cultured hepatocytes.

The binding and uptake of 59Fe-loaded 3H-labelled rat transferrin by cultured rat hepatocytes was investigated. At 4 degrees C, there is no evidence for a specific binding of transferrin which could be related to the association of neo-synthesized transferrin with plasma membrane receptors. At 37 degrees C, iron uptake is much more important than transferrin uptake; it proceeds linearly over the time of incubation, is largely proportional to the extracellular transferrin concentration, and is compatible with uptake by fluid phase endocytosis.

Receptor mediated endocytosis of hemoglobin-haptoglobin, galactosylated serum albumin and polymeric IgA by the liver.

Labelled hemoglobin-haptoglobin (ham-hap), galactosylated serum albumin (gal-SA) and polymeric immunoglobulin A (p IgA) were injected intravenously to rats or mice. The labels disappeared from the plasma with a half-time of about 5 min and were almost entirely found associated with the liver where degradation products progressively appear. The uptake of hem-hap and gal-SA are partially saturable as a function of the plasmatic concentration and the uptake of gal-SA can be completely inhibited by the simultaneous injection of asialofetuin.