Biomedical subjects

B Verrier

Publications and source records attributed to B Verrier.

At least 55 records · Page 3Linked to original sources

PubMed1992

Monocyte-independent T-cell activation by polyclonal antithymocyte globulins.

The in vitro mitogenic properties of polyclonal antithymocyte and antilymphocyte globulins (ATG) on peripheral blood mononuclear cells were investigated. The ATG were mitogenic in a dose-dependent manner with maximal proliferation observed at 250 or 500 micrograms/ml. ATG activated blastogenesis of CD4+, CD8+, and CD57+ (NK cells) lymphocytes. The ATG induced interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) gene expression and lymphokine secretion in cell culture supernatant and IL-2 receptor (CD25) expression. At submitogenic concentrations ATG potentialized the effect of phorbol esters on T cell proliferation, but not that of calcium ionophore. The mitogenic effect of ATG was not abrogated by monocyte depletion indicating that like CD2 monoclonal antibodies (mAbs) ATG activate T cells via a monocyte-independent pathway. CD3 and CD2 mAbs which activate T cells without binding to B surface determinants stimulated the proliferation of B cells and their differentiation into immunoglobulin (Ig)-secreting cells. In contrast, ATG induced only a transient B cell activation, but failed to support B cell differentiation into Ig-secreting cells despite the secretion of IL-2. These properties shared by ATG obtained in horses or rabbits by immunization with different cell types appear to differ from those of other T cell mitogens.

Antibody Formation↗

PubMed1992

Second signal for T lymphocyte activation: multiple targets for pharmacological modulation.

Complete T cell activation requires at least two signals. The first is delivered through the antigen-specific T cell receptor, whereas the second is generated by cognate interactions through adhesion molecules of T cells and antigen-presenting cells and/or by cytokines produced by antigen-presenting cells. The delivery of the two signals results in gene transcription, cytokine secretion, expression of new cell surface molecules including cytokine receptors, and cellular proliferation. Reference immunosuppressive agents were shown to act at different subsequent stages of T cell activation and proliferation. Among immunostimulating compounds, those which can enhance T cell responses are likely to modulate or replace the second signal in T cell activation, or alternatively, to act at later stages such as cytokine secretion, cytokine receptor expression or response to cytokine signals. For the purpose of in vitro evaluation of the activity of immunomodulators on the second signal of T cell activation, we devised a model of accessory cell depletion and reconstitution. This model allows the capacity of a given compound to enhance surface adhesion molecule expression or to trigger monocyte cytokine synthesis to be tested, and these effects to be assessed on T cell proliferation.

Adjuvants, Immunologic↗

PubMed1992

Inhibition of CD25 (IL-2R alpha) expression and T-cell proliferation by polyclonal anti-thymocyte globulins.

Anti-lymphocyte and anti-thymocyte globulins (ATG) are currently used as immunosuppressive agents in organ transplantation. Their administration in vivo may induce not only lymphocyte depletion but also functional effects which were investigated in the present study. In vitro ATG inhibited T-cell proliferation induced by monocyte-dependent T-cell mitogens, like CD3 antibodies, phytohaemagglutinin (PHA) and concanavalin A (Con A), by monocyte-independent mitogens, like CD2 antibodies, or by protein kinase C activators (phorbol esters) associated with a calcium ionophore. The inhibitory effect of ATG was therefore not solely accounted for by a suppression of co-stimulatory signals delivered by monocytes, but rather implied a direct action on T cells. Addition of recombinant human interleukin-2 (rIL-2) did not overcome the inhibition. Suppression of T-cell proliferation by ATG was characterized by normal RNA synthesis and IL-2 secretion contrasting with markedly reduced expression of the CD25 protein [p55, the alpha-chain of interleukin-2 receptor (IL-2R)] both in cytoplasm and on T-cell membrane, as well as a decreased secretion of interferon-gamma (IFN-gamma). Northern blot analysis revealed increased levels of CD25 and IFN-gamma mRNA, suggesting a post-transcriptional inhibition of these molecules, whereas IL-2 mRNA levels were unchanged. These data demonstrate that inhibition of T-cell proliferation by ATG can be attributed primarily to a post-transcriptional defect of CD25 expression, implying a novel mechanism different from those described with other immunosuppressive agents. Blocking of T-cell proliferation in the late G1 phase of the cell cycle may contribute to the immunosuppressive activity of ATG in prophylactic treatment of allograft rejection.

Antigen-Presenting Cells↗

PubMed1992

[Suramin induces lysosomal storage disorder in HT29-D4 cells during a mechanism of polarized endocytosis].

During the course of suramin-induced differentiation, a marked lysosomal storage disorder is observed in HT29-D4. This impairment could account for the toxic side effects of the drug during clinical trials in humans. It is shown that the perturbation is caused by a process of endocytosis of suramin-serum albumin complexes by the apical membrane of differentiated HT29-D4 cells.

Adenocarcinoma↗

PubMed1991

Kinetics of biochemical, electrophysiological and morphological events (including lysosomal disorder) during the course of suramin-induced differentiation of the human colon-cancer cell clone HT29-D4.

Suramin, a polysulfonated naphtylurea, has been shown to bind to a wide variety of tumor growth factors, and to exhibit anti-proliferative effects on several cell lines. We have followed the suramin-induced (100 micrograms/ml) evolution of morphological, biochemical and electrophysiological changes in HT29-D4 human colonic adenocarcinoma cell clone as a function of culture time. After 5 days of culture in the presence of the drug the cells were polarized and exhibited apical brush border and tight junctions. The polarization process of carcino-embryonic antigen (CEA) in the apical membrane domain was achieved after 8 days of treatment, while the correct localization of HLA class-I molecules in the basolateral membrane domain occurred after 14 days of culture in the presence of suramin. Spontaneous potential difference (PD) and transepithelial resistance (Rt) were recorded from the 9th day of treatment and reached maximum values at day 15 (PD = 3 mV; RT = 450 omega cm2), giving evidence that the differentiation process triggered by suramin concerned virtually all cells in the monolayer. Untreated cells were consistently found to be electrically inactive. Finally, from day 10 of suramin treatment, the lysosomal system was perturbed including accumulation of large autophagic vacuoles and, later, typical lamellar inclusion bodies. These structures were never seen when cells were induced to differentiate in suramin-containing serum-free medium. Moreover, similar perturbations of the lysosomal system could be obtained by adding BSA in the suramin-containing defined medium, suggesting that the lysosomal storage disorder occurring upon suramin treatment was due to endocytosis of suramin-BSA complexes. We conclude that lysosomal impairment due to the presence of BSA in the culture medium did not prevent HT29-D4 cells from differentiating and that it was not an early event which could be involved in the mechanism of action of suramin. However, this perturbation might account for some of the toxic effects occurring during chronic suramin treatment in humans.

Cell Differentiation↗

PubMed1991

A voltage, calcium, and ATP sensitive non selective cation channel in human colonic tumor cells.

A non selective cation channel has been identified in the human colonic cell lines T84 and HT29D4 using the patch clamp technique. The channel is equally permeable to Na+ and K+, has a linear current-voltage relationship and a conductance of about 20 pS in symmetrical NaCl conditions. The channel is not permeable to chloride or to large organic cations such as N-methyl-D-glucamine. The open probability of the channel is voltage dependent. Cytosolic Ca2+ concentrations higher than 0.1 mM are required to activate the channel. The channel is blocked by cytosolic ATP (1 mM). 3',5-dichlorodiphenylamine-2-carboxylic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid inhibit the channel when present on the extracellular side. The block is not voltage dependent. 3',5-dichlorodiphenylamine-2-carboxylic acid is the most potent blocker and completely inhibits channel activity at a concentration of 50 microM. The channel is insensitive to amiloride and derivatives.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Combination of culture on collagen gels and glucose starvation for cloning human colon cancer cells. Obtention of clones exhibiting different patterns of enterocytic differentiation.

Glucose starvation has been widely used to select differentiated subpopulations from the heterogenous human colon cancer cell line HT29. We observed that the important cell loss elicited by culturing these cells in glucose-free medium could be limited when type I collagen gel was used as substratum instead of conventional plastic support. We took advantage of this property to develop a new protocol, which combined glucose starvation and culture on collagen gels, for cloning HT29 cells. Using this procedure we have isolated four clones that were characterized on the basis of morphological (optical and transmission electron microscopy), electrophysiological (determination of transepithelial electrical parameters) and biochemical (detection of villin, sucrase-isomaltase and carcinoembryonic antigen) criteria. These four clones expressed different patterns of enterocytic differentiation regarding to these criteria. These results confirmed the heterogeneity of the HT29 cell line. One of these clones, HT29-A7, which displayed numerous intercellular cysts that disappeared at confluency, appears as a complementary model in the study of epithelial biogenesis.

Cell Differentiation↗

PubMed1991

Vectorial release of carcinoembryonic antigen induced by IFN-gamma in human colon cancer cells cultured in serum-free medium.

Confluent monolayers of intestinal cell lines are useful models for studies of intestinal epithelial structure and function. Three cell lines have retained morphological and functional properties of intestinal epithelial cells compatible with such studies: Caco-2, T84 and HT29. However, the requirement of fetal bovine serum for the culture of these cells does not facilitate the design of experiments dealing with growth factors or hormonal regulation. The clonal intestinal cell line HT29-D4 can be cultured as fully differentiated epithelial monolayers in a synthetic medium containing transferrin, selenous acid, epidermal growth factor and suramin, a potent differentiation inducer. In the present study it is shown that HT29-D4 cells grown on permeable substratum in this synthetic medium developed electrically active monolayers consisting of columnar cells with morphological characteristics of normal enterocytes. After metabolic labelling with [35S]-methionine, HT29-D4 monolayers released most of their radiolabelled secretory proteins preferentially in the basal compartment of the cell culture chamber. However, the carcinoembryonic antigen, shown to be present in the apical plasma membrane, was exclusively released apically. This oriented release was stimulated by recombinant gamma-interferon (IFN-gamma) added only in the basal chamber, suggesting a basolateral restriction for IFN-gamma receptors.

Blotting, Western↗

PubMed1991

Immunoreactive and bioactive isoforms of human thyrotropin.

Isoforms of intrapituitary human TSH were separated by gel isoelectrofocusing, and their immunoreactivity analyzed by subsequent immunoblotting using polyclonal and monoclonal antibodies. Under these conditions, TSH polymorphism could be resolved as seven major isoforms (pI 8.6, 8.3, 8.0, 7.5, 7.0, 6.5, and 6.0) by both silver staining of the gels and binding to anti-TSH polyclonal antibodies. The distribution pattern of these forms appeared totally distinct from that of individual TSH alpha (pI 8.8, 8.4, 8.2, 7.6, 7.4, 6.8, 6.6, 5.8, and 5.4) and TSH beta (pI 8.7, 8.1, 7.2, 6.8, 6.2, and 5.8) subunits. While most anti-TSH polyclonal antibodies recognized neutral and alkaline isoforms of TSH (pI 8.6, 8.3, 8.0, 7.5, 7.0, 6.5, and 6.0) through beta determinants, they displayed a variable potency to bind acidic forms of the hormone (pI 5.8, 5.5, 4.8, and 4.5), in contrast to anti-TSH alpha antisera, which enlighted the broadest spectrum of isoforms. Monoclonal antibodies of various specificities largely reproduced this distribution, indicating that at least five distinct epitopes are coexpressed in the neutral and alkaline forms of TSH, but only two are expressed in the acidic ones. All of the forms were found to induce cAMP production and stimulate growth of FRTL-5 rat thyroid cells, although neutral forms proved to be definitely less potent than the others. We therefore, conclude that TSH isoforms differ in the expression of both their immunoreactive and bioactive domains and that the bioactive/immunoreactive ratio is not an accurate index for the biopotency of the hormone.

Antibodies↗

PubMed1991

Retrovirus-mediated gene transfer of a human c-fos cDNA into mouse bone marrow stromal cells.

A cDNA encoding a complete human c-fos protein was isolated and inserted into two different murine MoMuLV-derived recombinant retroviruses allowing expression of c-fos protein in different cell types. One c-fos-expressing retrovirus, chosen for its ability to express high levels of proteins in fibroblast-like cells, was shown to potentiate long-term cultures of mouse bone marrow stromal cells in vitro and therefore constitutes a potential tool for immortalizing such cells. Moreover, when tested in an in vitro differentiation assay, stromal cells constitutively expressing c-fos favor the granulocyte differentiation of hematopoietic precursors. Interestingly, retroviruses expressing v-src and v-abl oncogenes, included as controls in our experiments, do not produce any detectable effects, whereas those expressing polyoma virus middle T antigen facilitate long-term growth in vitro of stromal cells that favor the macrophage differentiation pathway of bone marrow stem cells. Our observation supports the idea that constitutive expression of some oncogenes, including c-fos and polyoma virus middle T antigen, may influence cytokine production by bone marrow stromal cells.

Animals↗

PubMed1991

Four regulatory elements in the human c-fos promoter mediate transactivation by HTLV-1 Tax protein.

Expression of the human c-fos proto-oncogene is activated in trans by the Tax protein encoded by human T-cell leukemia virus type-1 (HTLV-1). Indeed, we show here that a HeLa clone stably transfected by Tax expresses Fos at a high level. We also show that multiple elements of the human c-fos promoter, i.e. the v-sis conditioned medium inducible element (SIE), the dyad symmetry element (DSE) necessary for growth factor induction, the octanucleotide direct repeat element (DR), and the cyclic AMP response element (CRE) centred at -60, can all mediate Tax transactivation. In the DSE, the 10bp central core that binds the serum response factor (SRF) is, by itself, sufficient to mediate Tax transactivation. Moreover, a CRE-binding protein is involved in Tax activation through the CRE-60 element. Since Fos is a transregulator of cellular genes, our results suggest that the oncoprotein plays a crucial role in T-cell transformation by HTLV-1 in conjunction with other Tax-inducible genes.

Base Sequence↗

PubMed1991

Transactivation of Krox-20 and Krox-24 promoters by the HTLV-1 Tax protein through common regulatory elements.

The HTLV-1 Tax protein has been shown to induce the expression of host cellular genes, some of which play crucial roles in cell proliferation and differentiation. We have examined the effect of Tax on the expression of two immediate-early genes, Krox-20 and Krox-24, which encode transcription factors. Several HTLV-1-infected T-cell lines and a HeLa cell line that constitutively expresses the Tax protein have a high level of expression of the Krox-20 and Krox-24 genes. In addition, Tax transactivates the promoters of both Krox-20 and Knox-24 in a co-transfection assay. Tax-responsive elements in Krox-20 and Krox-24 include the serum response elements (SREs) and the putative cAMP-responsive element (CRE). A correlation exists between the ability of these elements to mediate Tax transactivation and their affinity for their cognate factors, the serum response factor (SRF) or the CRE-binding protein (CREB) respectively. Since Tax is also able to transactivate the human c-fos promoter through the SRE and the CRE-60, our findings support the idea that the HTLV-1 Tax protein uses common mechanisms for transactivation of these three immediate-early genes. Deregulation of their expression may contribute to malignant transformation associated with HTLV-1 infection.

Base Sequence↗

PubMed1990

Ca2+ channel blockers inhibit secretory C1-channels in intestinal epithelial cells.

Outwardly rectifying Cl- channels are present in the human colonic cell line (HT29D4). The classical Cl- channel blocker 5-nitro-2(3-phenylpropylamino)benzoate inhibits Cl- channel activity with a K0.5 value of 20 microM. Epithelial Cl- channel activity is inhibited by Ca2+ channel blockers. Phenylalkylamines are the most effective inhibitors. (+/-)Verapamil and (-)desmethoxyverapamil induce flickering and then the complete blockade of Cl- channels recorded from outside-out patches. K0.5 values are 60 microM and 100 microM for (-)desmethoxyverapamil and (+/-)verapamil, respectively. Other classes of L-type Ca2+ channel blockers have also been studied but they are less active.

Adenocarcinoma↗

PubMed1990

Small conductance chloride channels in the apical membrane of thyroid cells.

A small conductance chloride channel has been identified on the apical membrane of porcine thyroid cells using the patch-clamp technique. In cell attached membrane patches with NaCl in the pipette, the single channel conductance is 5.5 pS. The channel is highly selective for chloride over gluconate and iodide, and is impermeable to Na+, K+ and tetraethylammonium ions. The open state probability of the channel is not affected by voltage. The channel activity disappears after excision of the patch. The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) did not affect the activity of the thyroid Cl- channels. Treatment of thyroid cells with 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (8-chloro-cAMP) (0.5 mM) prior to giga-seal formation increased Cl- channel activity in the apical membrane of thyroid cells.

Animals↗

PubMed1990

Evidence for probenecid-sensitive organic anion transporters on polarized thyroid cells in culture.

Epithelial thyroid cells in primary cultures loaded with BCECF/AM rapidly released the impermeant fluorescent dye BCECF (bis(carboxyethyl)carboxyfluorescein) in the incubation medium. Cells organized into follicles rapidly cleared BCECF (80% within 10 min) whereas fluorescence microscopy did not show any fluorescence in the follicular cavity. Cells organized into monolayers on plastic exported BCECF into the medium (70% within 40 min) whereas fluorescence microscopy showed intense fluorescence under the domes. BCECF efflux was blocked by probenecid, one of the known inhibitors of organic anion transporters, with similar efficiency in both structures. Maximal and half-maximal effects were respectively observed for 5 mM and 0.4 mM probenecid. The polarity of BCECF efflux was studied by using monolayers on collagen-coated Nuclepore filters: 85% of BCECF released was found in the basal compartment and 15% in the apical compartment. These findings suggested that thyroid cells in culture expressed a transport mechanism for the anionic form of BCECF. Furthermore, the observed activation of the Na+/H+ exchanger by probenecid suggested that the presence of this blocker did not overcome problems arising in the use of BCECF as intracellular pH indicator for thyroid cells.

Ammonium Chloride↗

PubMed1990

Suramin-induced differentiation of the human colic adenocarcinoma cell clone HT29-D4 in serum-free medium.

The clonal cell line HT29-D4 was able to grow in a completely defined medium containing EGF, selenous acid, and transferrin in the presence of the anti-helminthic drug suramin. In the absence of suramin, the kinetics of cell growth and the cell density obtained were dependent on the external EGF concentration. In the presence of suramin, cell density reached a plateau independent of EGF concentration above 50 ng/ml. At the morphological level, suramin allowed hemicyst formation in the cell monolayer. The cells were polarized with a well-ordered brush border facing the culture medium and mature junctional complexes that divided the cell membrane in two distinct domains. The carcinoembryonic antigen was found to be restricted to the apical membrane domain while the major histocompatibility molecules HLA-ABC were segregated within the basolateral domain. The electrical parameters of suramin-treated cells grown on permeable filters were measured and demonstrated that the cell monolayer was electrically active. These properties were never found in the absence of the drug. Moreover, the vasoactive intestinal polypeptide (VIP) was able to induce a dramatic increase in cAMP only when it was added, in agreement with the localization of the VIP receptor, in the lower compartment of the culture chamber. In conclusion we described for the first time conditions allowing the growth of functionally differentiated human colic cell monolayers in chemically defined medium. This model will contribute to a better understanding of suramin action and of the mechanisms involved in cell polarization.

Adenocarcinoma↗

PubMed1990

High levels of c-fos proto-oncogene expression in normal human adult skin.

The proto-oncogene c-fos is thought to play an important role in the modulation of cell growth and differentiation. In normal tissues that have been studied to date, c-fos expression has been found to be regulated in a tissue-specific manner. Actually, little is known about its expression in normal human adult skin (NHAS). Moreover, the epidermis is a useful tissue to study the role of cellular oncogenes because keratinocytes can be observed simultaneously in their proliferative as well as differentiated state. We studied c-fos expression in NHAS using different molecular approaches which permit us to characterize and localize c-fos products within the epidermis, specifically, at the RNA level by Northern blot and in situ hybridization, and at the protein level by immunofluorescence and Western blotting. Here, we show that both c-fos mRNA and protein are present at high levels in NHAS. These results contrast with the low level of c-fos expression reported for most human adult tissues. Furthermore, c-fos expression is visible throughout the epidermal layers indicating that it is not restricted to proliferating basal cells. The epidermis, therefore, represents the first human adult tissue where c-fos is expressed at high levels in vivo and provides an interesting model to further elucidate the role of this proto-oncogene in normal and pathologic conditions.

Blotting, Northern↗

PubMed1990

Products of the fos and jun proto-oncogenes bind cooperatively to the AP1 DNA recognition sequence.

The products of the proto-oncogenes c-fos and c-jun form a tight protein complex that is a major component of the transcription factor AP1. To analyze the role of fos in the binding of this complex to the AP1 DNA recognition sequence and the mechanism of interaction in further detail, we have expressed a fos protein in E. coli using an expression vector containing the temperature-inducible lambda PL promoter and a synthetic translational start codon. The fos protein encoded by this construct (termed Baf) was enriched by biochemical purification techniques and was found to form a specific complex with c-jun obtained by in vitro transcription/translation. As shown in gel retardation assays, the baf/jun complex binds to the AP1 DNA recognition sequence with high affinity, while no significant binding was observed with either of the individual protein components, indicating cooperative DNA binding of the two proteins. The fact that the bacterial baf protein does not undergo glycosylation indicates that the post-translational modification of eukaryotic c-fos with N-acetylglucosamine is not required for the formation of a stable fos/jun/DNA complex.

Binding Sites↗