Biomedical subjects

J Uriel

Publications and source records attributed to J Uriel.

At least 37 records · Page 2Linked to original sources

PubMed1992

Endogenous and exogenous alpha-fetoproteins as differential markers of cultured neonatal mouse Schwann cells and fibroblasts.

alpha-Fetoprotein (AFP) and AFP-gene transcripts were demonstrated in vitro in Schwann (S) cell and fibroblast (F) cell cultures of neonatal mouse origin. All S and F cells of primary cultures and of established cell lines expressed the AFP gene. AFP mRNA was detected by an in situ hybridization technique using a 35S-AFP-cDNA probe. AFP was localized by immunocytoperoxidase labelling using purified anti-AFP antibodies. The amounts of stained endogenous AFP, estimated semiquantitatively, were about 3-fold higher in S cells than in F cells. After incubating the cultures with exogenous mouse AFP, both S and F cells showed significant ability to take up the protein; the amount of internalized protein was found to be higher in F cells than in S cells. Moreover, the uptake of AFP fluorescein conjugates (FITC-AFP), estimated quantitatively by fluorometry, also gave higher values for F cells. The cytoplasm of F cells exhibited a characteristic fluorescence pattern, strongly illuminated and dispersed grains; the cytoplasm of S cells was regularly labelled. If exogenous FITC-AFP uptake could be used to distinguish labelled F cells from S cells (with application for identification and selection of F cells), the immunocytochemically stained endogenous AFP could allow S cells to be distinguished from F cells (using the dilutions of antibodies still staining the S cells but which lead to the absence of F-cell labelling). The two procedures, which can be used independently or together, may constitute differential markers for S cell and F cultures in, i.e., nerve regeneration of neurofibroma studies using the model of mixed S and F culture also containing other types of cells.

Animals↗

PubMed1992

Increased cytotoxicity of polyunsaturated fatty acids on human tumoral B and T-cell lines compared with normal lymphocytes.

Epidemiological and experimental data suggest that fatty acids may modulate the growth of tumor cells. We have analyzed the effect of different types of fatty acids, bound to serum proteins in physiological conditions, on the lipid composition and growth of human neoplastic B and T-cell lines and compared their effect on normal lymphocyte proliferation. Fatty acids with 0 to 2 unsaturations (stearic, oleic, and linoleic), at concentrations up to 50 or 100 microM did not significantly affect the proliferation of leukemic cells. However, long-chain polyunsaturated fatty acids (PUFA), and mainly docosahexaenoic (22:6, n-3), were cytotoxic at concentrations greater than or equal to 20 microM after 48-72 h in culture. Simultaneous supplementation with vitamin E restored normal cell growth. The amount of end-products of lipid peroxidation in cells correlated with the observed toxicity but the amount of superoxides did not. Fatty acid supplementations increased cell triacylglycerol content but did not affect the degree of unsaturation of phospholipids, cholesterol/phospholipids molar ratio, or membrane fluidity. Glutathione-S-transferase activity was low in Raji and CEM cells, moderate in lymphocytes and high in Ramos cells and did not increase with supplementations. The proliferation of normal lymphocytes, which produced lower amounts of end-products of lipid perodixation, was not inhibited, but in some cases stimulated, by PUFA (with the exception of 30 microM 22:6). The extension of these results to situations in vivo could lead to use of PUFA for delaying leukemia progression or in adjuvant chemotherapy.

Burkitt Lymphoma↗

PubMed1992

Activated human T lymphocytes express albumin binding proteins which cross-react with alpha-fetoprotein.

The kinetics of iodinated human serum albumin ([125I]Hu-SA) and alpha-fetoprotein ([125I]Hu-AFP) binding and endocytosis by resting and phytohemagglutinin (PHA)-activated human T lymphocytes were studied comparatively. The binding of both SA and AFP appeared considerably increased upon blastic transformation. SA, like AFP, binds in a saturable way to the surface of PHA-stimulated human T lymphocytes at 4 degrees C and is endocytosed at 37 degrees C. Two saturation plateaus were observed by incubating at 4 degrees C activated T lymphocytes with [125I]Hu-AFP at different concentrations (10 ng-250 micrograms/ml), while only one saturation plateau was obtained by incubating cells with [125I]Hu-SA in the same conditions. Scatchard analysis of binding data revealed two types of binding sites for Hu-AFP and one for Hu-SA. Competition experiments using proteins of human and bovine origin are in favor of the presence on the surface of these cells of a common binding site for AFP and SA. Pulse-chase experiments showed that internalized [125I]SA was released mainly in a degraded form from the cells, in agreement with detection by ultrastructural cytochemistry of peroxidase-conjugated SA in lysosome-like bodies by ultrastructural cytochemistry. This contrasts with the intracellular pathway of AFP, which as previously described (Geuskens, M., et al., Eur. J. Cell Biol. 50, 418-427 (1989)), moves to tubular-vesicular structures in the Golgi region and is recycled for the most part undegraded.

Albumins↗

PubMed1991

Activation of an alpha-fetoprotein (AFP)/receptor autocrine loop in HT-29 human colon carcinoma cells.

Immunological and morphological approaches have been used to demonstrate, respectively, alpha-fetoprotein (AFP) synthesis and receptor expression in the HT-29 human colon carcinoma parental cell line. HT-29 cells cultivated in the presence of glucose synthesized and secreted AFP in the medium from 48 to 96 hr after seeding, as revealed by monoclonal antibodies (MAbs) to human AFP. Light microscopic observations of cells incubated with fluoresceinated AFP showed that the protein was specifically bound to the cell surface at 4 degrees C, and was internalized in the cytoplasm at 37 degrees C. At the ultrastructural level, horseradish peroxidase (HRP)-conjugated AFP, as well as HRP-transferrin (Tf) (used as a control), appeared to be internalized via coated pits and vesicles before being delivered to endosomes, from which they were apparently recycled back to the cell surface via small vesicles. Our results suggest that a AFP/receptor autocrine pathway might operate in these cells, be preferentially active at the beginning of the exponential phase of cell culture growth, and contribute to cell proliferation.

Colonic Neoplasms↗

PubMed1991

Receptor-mediated endocytosis and recycling of alpha-fetoprotein in human B-lymphoma and T-leukemia cells.

The kinetics of iodinated human alpha-fetoprotein (AFP) binding and uptake by 2 human neoplastic lymphoid cell lines (CEM and RAJI) have been studied. Three saturation plateaus were obtained by incubating CEM and RAJI cells at 4 degrees C with 125I-AFP at different concentrations. Scatchard analysis suggested the presence of 3 types of receptor site with different affinities and capacities on cells of both lines. AFP binding was inhibited by unlabelled human and bovine AFP, and to a lesser extent by human serum albumin (SAH); no significant competition was observed with human transferrin (Tf) or ovalbumin (Ova). Pulse-chase experiments showed that 125I-AFP was released practically undegraded from the cells. Covalent conjugates of AFP and Tf with horseradish peroxidase (HRP) were used to follow the endocytosis and intracellular pathway of these serum proteins by electron microscopy. Both proteins were observed in coated vesicles, endosomes and a tubular vesicular network localized in the Golgi-centrosphere region. SAH-HRP was internalized to a much lesser extent. Ova-HRP was poorly internalized and was observed in lysosome-like organelles.

Binding, Competitive↗

PubMed1991

Changes in lipid composition of human peripheral blood lymphocytes infected by HIV.

The possible differences in lipid composition between human immunodeficiency virus- (HIV) infected and uninfected PHA-activated human peripheral blood mononuclear cells (PBMC) have been studied. The total fatty acid composition was similar, except for the proportion of arachidonic acid, that was slightly higher in infected than in noninfected cells. No significant differences were obtained in the incorporation of radiolabeled stearic or oleic acids in the different lipid classes. The staining of cells with Nile Red showed similar amounts of intracytoplasmic lipid droplets. On the contrary, the CH/PL ratio, the major factor in determining cell membrane fluidity, was clearly higher in infected than in uninfected cells (0.60 and 0.36, respectively). This fact is discussed in relation with the known high CH/PL ratio (0.95) of the lipid envelope of HIV.

Cholesterol↗

PubMed1991

Alpha-fetoprotein uptake by differentiating neuroretinal structures of the chick embryo.

Internalization of exogenous fluoresceinated alpha-fetoprotein (FITC-AFP) was studied at different stages of development on embryonic chick neural retinas maintained, for short periods, in organ cultures. Cellular localization of endogenous, native AFP was carried out by immunohistoperoxidase methods. Cells which specifically internalized exogenous FITC-AFP (neurons and their processes) were precisely those showing positive immunolabeling for the endogenous, native protein. Such a result supports the hypothesis of a predominantly exogenous origin of intracellular neuroretinal AFP. A precise topography and temporal sequence of AFP labeling after internalization in retinal structures is given. AFP uptake was not displayed by undifferentiated cell precursors or germinal cell layers but was apparent in cells with phenotypic characteristics of maturing neurons. Nerve fibers and synaptic layers actively internalized FITC-AFP at specific stages of development. Fully differentiated neurons and processes did not internalize AFP. The possible role of AFP, a carrier of biologically active substances such as fatty acids, in neural retina differentiation is discussed.

Animals↗

PubMed1991

A study, by electron microscopy, of the specific uptake of alpha-fetoprotein by mouse embryonic fibroblasts in relation to in vitro aging, and by human mammary epithelial tumour cells in comparison with normal donors' cells.

A covalent conjugate of alpha-foetoprotein (AFP) and horseradish peroxidase (HRP) has been used to follow the internalization pathway of this serum protein in early and late passages of primary cultures of mouse embryonic fibroblasts as well as in a spontaneously immortalized cell line. AFP, as transferrin (Tf) used in parallel as a control, are endocytosed through coated pits and vesicles and move then to endosomes in every case; in cells of the late passages, at least a part of the internalized proteins would be routed to lysosomes. Cells of three different established human mammary cancer lines (MCF-7, Evsa-T, T-47D) internalize AFP-HRP through coated pits and vesicles. Such localization of the conjugate is practically never detected in normal human mammary epithelial cells in primary culture. Taken together, these results are in agreement with the view that AFP receptors are expressed at the surface of proliferating mouse embryonic fibroblasts and human mammary epithelial cancer cells but absent from the surface of normal human mature cells of the same origin.

Animals↗

PubMed1991

Quantification of alphafetoprotein (AFP) endocytosis by PHA-activated peripheral blood mononuclear cells (PBMC) from HIV-infected individuals. A useful test of predictive value for the progression of AIDS.

Serum alphafetoprotein (AFP) is actively taken up, through receptor-mediated endocytosis, by many embryo-fetal cells during ontogenic development but also by neoplastic cells, as well as by normal peripheral T lymphocytes after mitogenic activation. We have previously shown that the ability to internalize AFP is impaired in mitogen-activated T cells from several groups of (HIV+)-seropositive individuals and that this expression roughly correlates with the progression of the disease. It is not clear whether this impaired AFP-endocytosis results from an HIV-mediated inhibition of AFP-receptor expression or if it is the consequence of a target signal transduction impairment due to HIV-infection. In the present work we have explored both possibilities by studying AFP-endocytosis in peripheral blood mononuclear cells (PBMC) from seropositive (HIV+)-asymptomatic heterosexual individuals and in in vitro HIV-infected PBMC from healthy donors, either quiescent or stimulated with phytohemagglutin (PHA). Our results suggest that this novel abnormality of T-cells associated with HIV-infection reflects an unusual proliferative response of PBMC to mitogenic stimuli.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Quantification of alpha-fetoprotein and transferrin endocytosis by lymphoid cells using flow cytometry.

Alpha-fetoprotein (AFP) and transferrin (Tf) are serum proteins actively internalized by many growing cells through specific cell surface receptors. The intracellular pathways of AFP and Tf are very similar: both proteins enter the cells via coated pits and receptosomes, move to tubular elements of the transreticular portion of the Golgi and are recycled back to the cell surface and extracellular medium in native form. In the present work, the capacity of human lymphoid cells to bind AFP and Tf at 4 degrees C and to endocytose them at 37 degrees C was quantified by flow cytometry analysis on a FACS 440 using fluoresceinated derivatives of these proteins. The results obtained show that binding and internalization of AFP and Tf by lymphoid cells are saturable processes at either 4 degrees C or 37 degrees C. The method developed permits direct quantitative measurement of molecules bound to the cell surface or present within the cell.

Cell Line↗

PubMed1990

Cytotoxicity of chlorambucil and chlorambucil-fatty acid conjugates against human lymphomas and normal human peripheral blood lymphocytes.

The cytotoxic activity of chlorambucil (Chl) and of chlorambucil-fatty acid conjugates of different degree of unsaturation have been assayed in vitro upon two human lymphoma cell lines and comparatively, upon quiescent and mitogen-activated lymphocytes from healthy blood donors. The cell toxicity observed with Chl-arachidonic acid and Chl-docosahexaenoic acid against lymphoma cells was, at any experimental condition used, equal or higher than the individual toxic potential of either chlorambucil or fatty acids. The two conjugates, like chlorambucil alone, were toxic against mitogen-activated lymphocytes. Contrary to chlorambucil, Chl-arachidonic at any concentration tested, lacked of toxicity towards normal non-activated lymphocytes. Chl-oleic acid conjugate was, whatever the cell species tested, much less toxic than Chl alone. In conclusion, the coupling of chlorambucil with polyunsaturated fatty acids increases: (a) the selectivity against neoplastic versus quiescent lymphocytes and (b) the toxicity for B-lymphoma cells. The selective effect of Chl-fatty acid conjugates is discussed in relation with the expression of an AFP/AFP-receptor autocrine system in malignant lymphoblastoid cells and in mitogen-activated lymphocytes.

Arachidonic Acids↗

PubMed1990

Fatty acid metabolism in human lymphocytes. I. Time-course changes in fatty acid composition and membrane fluidity during blastic transformation of peripheral blood lymphocytes.

The time-course changes in fatty acid composition of human T-lymphocytes during blastic transformation were analysed, as well as the variations in membrane fluidity determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), using a fluorescence-activated cell sorter. The more important changes observed, in activated relative to quiescent cells, started after 24 h and consisted in an increase in the proportion of oleic (18:1(n - 9)), docosapentaenoic (22:5(n - 3)) and docosahexaenoic (22:6(n - 3)) acids and a decrease in that of linoleic (18:2(n - 6)) and arachidonic (20:4(n - 6)) acids. This represented a relative increase of 26% for 18:1, 56% for 22:5 and 84% for 22:6 in peripheral blood mononuclear cells (PBMC) and 35%, 182% and 94%, respectively, in purified T-lymphocytes, both activated for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and 19%, respectively, for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and phosphatidylethanolamine) rather than neutral lipids. The 18:1/18:0 ratio increased greatly in major cell phospholipids. The proportion of 20:4, 22:5 and 22:6 in phosphatidylinositol was not significantly altered after 72 h of activation. The molar ratio cholesterol/phospholipids was reduced in 72-h-activated lymphocytes (0.29) compared to quiescent cells (0.5). On the other hand, the stimulation of human T-lymphocytes caused a significant decrease in the order parameter (S) of DPH, according to the observed changes in lipid composition. After 72 h in culture, the S value for quiescent and stimulated T-lymphocytes was 0.530 and 0.326, respectively. In conclusion, the blastic transformation of human T-lymphocytes is associated with changes in lipid composition which modify the physical properties of their membranes. These modifications could modulate, in turn, the activity of membrane proteins implicated in the process of blastic transformation.

Cells, Cultured↗

PubMed1990

Fatty acid metabolism in human lymphocytes. II. Activation of fatty acid desaturase-elongase systems during blastic transformation.

The fatty acid desaturation-elongation ability of human T-lymphocytes during blastic transformation was determined both by gas-liquid chromatography and incubation with radiolabeled precursors. Human peripheral blood mononuclear cells (PBMC) were activated with phytohemagglutinin (PHA) and cultured in media supplemented with different fatty acids (18:0, 18:1(n - 9), 18:2(n - 6), 18:3(n - 3) and 20:4(n - 6)) at a final concentration of 30 microM. All the fatty acids added were elongated by activated PBMC and the maximal activity was observed on 20:4(n - 6) (a 25% of conversion to 22:4(n - 6)). Supplementation with stearic acid increased the proportion of oleic (from 21.4% to 23.7%) and eicosaenoic (from 3.1% to 5.7%) acids in cellular lipids, indicating the existence of a delta 9-desaturase activity. Supplementation with linoleic and linoleic acids increased slightly the cell content in their more unsaturated derivatives. Direct measurement of desaturase activities was performed by incubating quiescent and activated PBMC with [1-14C]stearic, [1-14C]linoleic and [1-14C]linolenic acids. Quiescent cells exhibited a very low delta 9-desaturase and no sign of delta 6-desaturase activity. A moderate and progressive activation of delta 9-, delta 6- and delta 5-desaturases was observed during blastic transformation of human PBMC. Up to 8% of 18:0 was converted to monoenes, 4% and 1.5% of 18:2(n - 6) was converted to trienes and tetraenes, respectively, and 14.5% of 18:3(n - 3) was converted to pentaenes. The maximal relative activities were found after 48 h of PHA-stimulation for delta 9-desaturase (around 90 pmol of 18:0 converted per 10(6) cells in the last 24 h) and at 72 h for delta 6- and delta 5-desaturases (around 75 and 140 pmol of 18:2 and 18:3, respectively, converted per 10(7) cells in the last 24 h). Although these activities are not enough to explain all the changes in fatty acid composition of human PBMC during blastic transformation, they may contribute to a more controlled cell phospholipid composition.

Acetyltransferases↗

PubMed1990

Defective uptake of alpha-fetoprotein (AFP) and transferrin (Tf) by PHA-activated peripheral blood lymphocytes from patients with AIDS and related syndromes.

Serum alpha-fetoprotein (AFP) and transferrin (Tf) are actively endocytosed by many growing cells during ontogenic and neoplastic growth, but also by peripheral T lymphocytes upon mitogen activation. AFP and Tf uptake occurs through receptor-mediated endocytosis. The purpose of the present work was to assess whether the expression and functional activity of AFP and Tf receptors are impaired in mitogen-activated T cells from several groups of HIV-1 seropositive (HIV+) individuals. Forty HIV+ cases were studied, including 12 patients with AIDS, 12 with lymphoadenopathy syndrome (LAS), as well as 16 asymptomatic homosexuals (As). Quantification of AFP and Tf uptake was carried out by fluorescence-activated cell sorting (FACS) using fluoresceinated derivatives of these proteins. Compared with healthy blood donors, the three HIV-1 seropositive groups exhibited clear impairment in the ability of their peripheral blood mononuclear cells (PBMC) to internalize AFP and Tf. The decrease in mean values of AFP uptake correlates roughly with the severity of the clinical status. Although these observations need to be confirmed after a much wider study groups, the AFP-Tf-endocytosis assay presented here clearly reveals early defective functions of mitogen-responsive T cells in disease-free subjects and may provide the basis for a prognostic test. The pathophysiological implications of these facts are discussed in relation to the structural and/or metabolic activities of fatty acids and iron, the ligands carried by AFP and Tf, respectively.

AIDS-Related Complex↗

PubMed1989

Alpha-fetoprotein gene expression in human lymphoblastoid cells and in PHA-stimulated normal T-lymphocytes.

Alpha-fetoprotein (AFP) is mainly synthesized by the fetal liver, the yolk sac and, to a much lower extent, by a few non-hepatic fetal tissues (i.e. kidney, pancreas, lung). This property is considered to be lost in mature quiescent cells of the adult. In the present we have studied the expression of AFP mRNA sequences in phytohemagglutinin (PHA)-stimulated normal human T-lymphocytes and in several human lymphoma cell lines. The amount of mRNA transcripts detected in quiescent T-lymphocytes by dot and Northern blot analysis was very low. It increased rapidly after PHA-activation, reached a maximum at 72 hours (six fold the level observed for quiescent T-lymphocytes) and decreased thereafter. The lymphoma cell lines Daudi, Raji, Rh6 et CEM, all expressed elevated levels of AFP mRNA. The transcripts had the size expected for human AFP, suggesting that they were functional and probably translated into protein. The possible role of AFP synthesis in lymphocyte blastogenesis and in lymphoma growth is discussed in relation with the strong binding affinity of this protein for polyunsaturated fatty acids.

DNA Probes↗

PubMed1989

Expression of alpha-fetoprotein receptors by human T-lymphocytes during blastic transformation.

Alpha-fetoprotein (AFP) and transferrin (Tf) are actively internalized by many growing cells during ontogenic and neoplastic development, including human malignant T- and B-lymphoblastoid cells. Their internalization is, on the contrary, greatly diminished or absent in mature, non-proliferating elements. In the present work, peripheral blood mononuclear cells (PBMCs) and T-lymphocytes, harvested from normal human donors, were induced to blastic transformation with phytohemagglutinin (PHA) and their ability to uptake AFP and Tf was measured and compared with Tf uptake in the same conditions. The capacity of the cells to internalize both proteins was quantified by fluorescence activated cell sorter (FACS) using fluoresceinated derivatives of these proteins. The results obtained show a significant uptake of AFP by T-lymphocytes upon PHA stimulation. The values of AFP incorporation were similar for all the cells studied (PBMCs, T-cells and T4, T8 cell subsets). The time course of AFP uptake paralleled, under the same conditions, the uptake of Tf and the expression of IL2 receptors. AFP uptake increased rapidly from the zero time (resting T-cells) and reached a maximum around 72 hr after PHA activation. Scatchard analysis of kinetic data at 4 degrees C revealed for Hu-AFP one single group of specific binding sites in PHA activated T-lymphocytes with a dissociation constant of 3.03 x 10(-7) M and around 88,000 sites/cell. There results strongly suggest the transitory expression of AFP receptors in T-lymphocytes during blastic transformation.

Cells, Cultured↗

PubMed1989

Alpha-fetoprotein and albumin uptake by mouse tissues during development.

We have studied the evolution of the incorporation of alpha-fetoprotein (AFP) and albumin by mouse tissues from fetal to adult life. Mice were injected with 125I-labelled AFP or albumin and, 3 h later, blood and organs were removed and analyzed for their radioactive protein content. Results showed that all immature tissues examined took up both AFP and albumin from blood. AFP uptake was higher during fetal and early postnatal life and decreased with age. In general, the relative uptake of albumin was lower than that of AFP, and the time-course incorporation of both proteins was parallel in brain, liver and kidney. In other tissues such as white adipose tissue, brown adipose tissue and skin. AFP uptake decreased while albumin uptake remained almost constant or increased with age. In the fetal period, the strong AFP uptake in white adipose tissue contrasted with the much lower albumin incorporation by this tissue. Autoradiographs from sections of organs and entire animals confirmed the cytoplasmic localization of AFP and albumin. We conclude that AFP and albumin found in developing tissues, except for fetal liver and yolk sac, proceed mostly from blood uptake. These results agree with recent experimental data suggesting that the major physiological role of AFP is the transport and delivery of polyunsaturated fatty acids to developing tissues.

Adipose Tissue↗

PubMed1989

Alpha-fetoprotein (AFP) in granulomatous inflammation of the mouse.

Inflammatory granulomas constituted of various proportions of macrophages, polynuclear (PMNs) and mononuclear cells were induced in adult pathogen-free mice by injecting polyacrylamide beads into subcutaneous pouches. Using specific anti-mouse alpha-fetoprotein (AFP) antibodies, the presence of AFP was immunocytochemically demonstrated in the cytoplasm of macrophages and of immature PMNs (mature PMNs and mononuclear cells were AFP negative). AFP-labelling started as soon as 36 h after granuloma induction and reached a maximum 60-72 h later, to disappear on day 5. The examination of different organs in these mice also showed a transitory labelling for AFP of liver hepatocytes and of elements of kidney and of exocrine pancreas. Parallel to these findings, the selective concentration inside the granulomatous pouches of radiolabelled AFP injected in the course of inflammation was observed. These results suggest that (a) shortly after the inflammatory reaction the synthesis of AFP is resumed by the liver; (b) the newly synthesized AFP is secreted in the serum and preferentially taken up by the granulomas; and (c) consequently, in adult mice, AFP behaves as a positive acute phase reactant. The physiopathological implications of these facts are discussed in relation with the biological properties of AFP.

Animals↗