Improvement of polyphenol properties upon glucosylation in a UV-induced skin cell ageing model.

OBJECTIVE: Polyphenols are strong antioxidant molecules allowing prevention of skin photo-ageing damages, but their use is limited due to low solubility and toxicity towards skin cells. We postulated that enzymatic glucosylation could improve their solubility, stability and, consequently, their efficacy. The aim of this work was to study changes induced by addition of a glucose moiety on two polyphenols displaying very different chemical structures [caffeic acid (CA), epigallocatechin-3-gallate (EGCG) and there glucosylated form, Glc-CA and Glc-EGCG] by assessing their cytotoxic properties and their antioxidant and anti-inflammatory activities. METHODS: Their antioxidant effect was assessed first by the classical DPPH radical-scavenging method. Then, a panel of human skin cells (keratinocytes, melanocytes, fibroblasts and endothelial cells) was used to evaluate their effect on cell toxicity and their antioxidant activities. With this aim, a photo-ageing model based on UV irradiation of skin cells was established. Molecule activity was assessed on reactive oxygen species (ROS) production, on superoxide dismutase (SOD) and catalase activities and, finally, on inflammatory factor production IL-6, IL-8 and IL-1ß. RESULTS: In an acellular model, antioxidant activity assessed by DPPH method was strongly reduced for Glc-CA compared to CA, whereas it remained the same for Glc-EGCG compared to EGCG. Glucosylated derivatives did not display more toxic effect on various skin cells. Moreover, toxicity was even strongly reduced for caffeic acid upon glucosylation. The efficacy of glucosyl-compounds against UV-induced ROS production was preserved, both with pre- and post-UV treatments. Particularly, a better antioxidant efficacy was shown by Glc-EGCG, vs. EGCG, on keratinocytes. In addition, an induction of SOD and catalase activity was clearly observed for Glc-CA. Both glucosyl-polyphenols display the same activity as their parent molecule in decreasing inflammatory factor production. CONCLUSION: Our results demonstrated that enzymatic glucosylation of CA and EGCG led to an improved or preserved antioxidant activity in a cellular model of UV-induced skin ageing, despite the decrease in instantaneous antioxidant properties observed for Glc-CA. Glc-EGCG is specifically more active on keratinocytes, suggesting a specific targeting. Such glucosylated polyphenols displaying improved physicochemical and biological properties should be better candidates than natural ones for use in food additives and cosmetics.

Induction of endothelial cell apoptosis by the antivascular agent 5,6-Dimethylxanthenone-4-acetic acid.

5,6-Dimethylxanthenone-4-acetic acid, synthesised in this laboratory, reduces tumour blood flow, both in mice and in patients on Phase I trial. We used TUNEL (TdT-mediated dUTP nick end labelling) assays to investigate whether apoptosis induction was involved in its antivascular effect. 5,6-Dimethylxanthenone-4-acetic acid induced dose-dependent apoptosis in vitro in HECPP murine endothelial cells in the absence of up-regulation of mRNA for tumour necrosis factor. Selective apoptosis of endothelial cells was detected in vivo in sections of Colon 38 tumours in mice within 30 min of administration of 5,6-Dimethylxanthenone-4-acetic acid (25 mg x kg(-1)). TUNEL staining intensified with time and after 3 h, necrosis of adjacent tumour tissue was observed. Apoptosis of central vessels in splenic white pulp was also detected in tumour-bearing mice but not in mice without tumours. Apoptosis was not observed in liver tissue. No apoptosis was observed with the inactive analogue 8-methylxanthenone-4-acetic acid. Positive TUNEL staining of tumour vascular endothelium was evident in one patient in a Phase I clinical trial, from a breast tumour biopsy taken 3 and 24 h after infusion of 5,6-Dimethylxanthenone-4-acetic acid (3.1 mg x m(-2)). Tumour necrosis and the production of tumour tumour necrosis factor were not observed. No apoptotic staining was seen in tumour biopsies taken from two other patients (doses of 3.7 and 4.9 mg x m(-2)). We conclude that 5,6-Dimethylxanthenone-4-acetic acid can induce vascular endothelial cell apoptosis in some murine and human tumours. The action is rapid and appears to be independent of tumour necrosis factor induction.

New human microvascular endothelial cell lines with specific adhesion molecules phenotypes.

Vascular endothelial cells recognize blood-borne circulating cells and allow them to extravasate in a tissue-specific manner. Because this property determines the selectivity of lymphocyte homing, it is fundamental in physiological as well as pathological processes (inflammation, autoimmune diseases, metastasis). As a tool to assess the molecular basis of endothelium selectivity, microvascular endothelial cell lines of distinct tissue origin were established. Endothelial cells, isolated from lymphoid tissues (lymph nodes and appendix) and from nonlymphoid immune sites--intestine, lung, and skin--were immortalized in vitro. Their general endothelial characteristics, such as the presence of von Willebrand factor (wWf), angiotensin-converting enzyme (ACE), VE-cadherin, and the intracellular E-selectin, were preserved. This article shows that these cell lines display phenotypic characteristics related to their tissue origin. Hence, endothelial cells from lymph nodes expressed peripheral lymph node addressins (PNAds). Endothelial cells from nonlymphoid tissues were ICAM-1 (intercellular adhesion molecule-1) and CD49e positive, whereas P-selectin was not equally distributed among the cell lines. Endothelial cells from mucosal sites reacted with antibody against human MAdCAM-1 (mucosal addressin cell adhesion molecule). In the adhesion test, lymphoid and myeloid cells adhere to endothelial cell lines in a distinct manner. These lines could be useful to study molecular mechanisms involved in tissue-specific cell-cell interaction.

Analysis of intracellular didanosine triphosphate at sub-ppb level using LC-MS/MS.

An analytical procedure has been developed for the analysis of intracellular didanosine triphosphate (ddATP). An electrospray ionization tandem mass spectrometer (ESI-MS) was interfaced to liquid chromatography (LC) using a mobile phase CH3OH/H2O (25/75) containing 1% formic acid for the analysis of the 5'-triphosphate metabolite of the antiviral didanosine. In this procedure, ddATP was extracted from CEM-T4 cells, isolated using an exchange anion solid phase extraction procedure, enzymatically dephosphorylated and then analyzed by LC-MS/MS within a 1 min run time. The influence of several parameters (electrospray ionization interface, acidic modifiers of the mobile phase) has been studied. A calibration curve was generated and the linear regression analysis yielded a regression coefficient (r(2)) greater than 0.999. Using LC-MS/MS detection in single reaction monitoring mode (SRM), the limit of quantitation of ddA in CEM-T4 cells was 0.02 ng ml(-1). Furthermore, this procedure could be used to perform simultaneous detection of five nucleoside reverse transcriptase inhibitors, such as AZT, 3TC, ddA, ddC and d4T and make LC-MS/MS a method of choice for Therapeutic Drug Monitoring (TDM) in a clinical environment.

Analysis of anti-HIV nucleoside inhibitors by capillary electrophoresis-electrospray ionization mass spectrometry.

A method employing capillary electrophoresis (CE) with tandem mass spectrometry (MS) has been developed for the simultaneous determination, on one hand, of zidovudine (AZT) with stavudine (d4T), and on the other hand, of lamivudine (3TC) with a didanosine metabolite (ddA), four potent human immunodeficiency virus reverse transcriptase (RT-HIV) inhibitors. The influence of several parameters (pH and ionic strength of volatile formic acid-ammonia buffer) as well as the influence of magnesium cation upon electroosmotic flow, electrophoretic mobility and peak efficiency has been studied. The limit of detection (LOD) by this method is 2.5 ppb for AZT and 20 ppb for d4T, 2 ppb for ddA and 5 ppb for 3TC, respectively. This paper illustrates the current importance in CE-ESI/MS/MS technique as a complementary or substituted method to measure levels (at ng/mL) of anti-HIV drugs alone or in combination.

KDR promoter can transcriptionally target cytosine deaminase suicide gene to cancer cells of nonendothelial origin.

The KDR/flk-1 gene promoter is considered to be endothelial cell-specific. We show its activity in two cancer cell lines of non-endothelial origin: in murine L1 sarcoma and OVP-10 human ovarian carcinoma cell lines. KDR promoter-driven cytosine deaminase gene can be efficiently expressed in these cells leading to sensitization to 5-fluorocytosine, as demonstrated both in vitro and in vivo. Our results indicated that KDR promoter activity is not endothelial cell-exclusive and that this promoter can also be used to obtain specific expression of therapeutic genes in certain cancer cells.

Receptor-mediated transcytosis of cyclophilin B through the blood-brain barrier.

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein mainly located in intracellular vesicles and secreted in biological fluids. In previous works, we demonstrated that CyPB interacts with T lymphocytes and enhances in vitro cellular incorporation and activity of CsA. In addition to its immunosuppressive activity, CsA is able to promote regeneration of damaged peripheral nerves. However, the crossing of the drug from plasma to neural tissue is restricted by the relative impermeability of the blood-brain barrier. To know whether CyPB might also participate in the delivery of CsA into the brain, we have analyzed the interactions of CyPB with brain capillary endothelial cells. First, we demonstrated that CyPB binds to two types of binding sites present at the surface of capillary endothelial cells from various species of tissues. The first type of binding sites (K(D) = 300 nM; number of sites = 3 x 10(6)) is related to interactions with negatively charged compounds such as proteoglycans. The second type of binding sites, approximately 50,000 per cell, exhibits a higher affinity for CyPB (K(D) = 15 nM) and is involved in an endocytosis process, indicating it might correspond to a functional receptor. Finally, the use of an in vitro model of blood-brain barrier allowed us to demonstrate that CyPB is transcytosed by a receptor-mediated pathway (flux = 16.5 fmol/cm2/h). In these conditions, CyPB did not significantly modify the passage of CsA, indicating that it is unlikely to provide a pathway for CsA brain delivery.

Role of lectin-glycoconjugate recognitions in cell-cell interactions leading to tissue invasion.

Endogenous lectins are cell receptors, expressed in normal and transformed cells both circulating as well as in organized tissues. Their biological significance was shown in developmentally regulated processes of cell migration, embryonic maturation, differentiation and during various other normal and pathological processes. This work will focus on the role of endogenous lectins and their glycoconjugate ligands in homing of circulating normal and cancer cells. During the normal immune process of lymphocyte recirculation and their journey among the whole body through the secondary lymphoid organs in the search for antigen, lectins are decisive molecules that allow the very first interaction of arrest onto the endothelial cell layer. It has been demonstrated that dual lectin-glycoconjugate interactions were taking part in the initiation of the whole adhesion cascade between adhering and endothelial cell. This indicates the role of the endothelium which will be described here. Indeed, using high endothelial cell lines immortalized by us, we could demonstrate that endothelium of microcapillaries is characterized by its tissue-specific properties although with a high microenvironment dependency. Both are decisive for selecting cells to stop and undergo further invasion. Such normal properties of endothelial cells and homing cells could be taken as example to understand pathologies like specific establishment of metastases in the case of cancer cells. Consequently, we shall take into account the potential offered by lectins and the knowledge of the structure of their ligand to design efficient adhesion blockers or enhancers as invasion inhibitors or immunomodulators.

Ultraviolet rays induced expression of lectins on the surface of a squamous carcinoma keratinocyte cell line.

Human keratinocytic cells from squamous carcinoma (SCL-1) present, under resting conditions, relatively low amounts of endogenous lectins (sugar-binding proteins). Upon uv irradiation, they express on their cell surface large amounts of endogenous lectin molecules able to bind neoglycoproteins bearing either alpha-L-rhamnosyl or alpha-D-glucosyl residues. A similar binding specificity was found with normal human keratinocytes under the same culture conditions. At sunlike doses, uv.A (365 nm) was more efficient than uv.B (312 nm) in the expression of such receptors on the surface of SCL-1 cells. The increased presentation of lectins by SCL-1 cells was transient and reached a maximum 4 h after irradiation. Such a specific modulation of receptor expression upon uv irradiation might be biologically significant, considering the numerous intercellular recognition phenomena in skin biology. alpha-L-Rhamnose-specific receptor on SCL-1 could not be distinguished from alpha-D-glucose-specific receptor on the basis of neoglycoproteins binding, uptake, and related inhibitions. Lectin expression was mainly detected on the cell surface, and its overexpression due to uv rays required a de novo protein synthesis process.

Selective induction of peripheral and mucosal endothelial cell addressins with peripheral lymph nodes and Peyer's patch cell-conditioned media.

Vascular endothelial cell addressins play an important role in lymphocyte homing in secondary lymphoid organs and in chronic inflammatory areas. A SV40 large T antigen-immortalized cell line from peripheral lymph nodes, HECa1O [Bizouarne et al., 1993a], was used to characterize the location of addressins with regard to environmental factors and cytokines. For this purpose, two monoclonal antibodies, MECA 79 and MECA 367, specific for peripheral lymph node vascular addressin and for mucosal addressin (Peyer's patches), respectively, were bound to unstimulated HECa1O cells. Both mucosal and peripheral addressins were detected inside the cells and in cellular extracts of the resting cells. On the cell surface, both addressins could be evidenced on the same cells at a moderate level of expression. They partly mediate the EL4/EL4IL2 lymphoma cells' adhesion to HECa1O cells. Supernatants of cultured peripheral lymph node or Peyers' patch cells induced expression of MECA 79 or MECA 367 antigens, respectively, on the surface of HECa1O cells. Interleukins, IL-7, IL-3, and IL-8, induced the cell-surface appearance of MECA 79 but not of MECA 367 antigen. Therefore, the same cell type synthesizes both antigens, but the expression of these antigens on the cell surface is independently regulated, thus uncovering a characteristic tissue type-specific as well as environment-sensitive properties of microvascular endothelial cells.

The immunoaugmenting properties of murine IgD reside in its C delta 1 and C delta 3 regions: potential role for IgD-associated glycans.

IgD receptor (IgD-R) bearing CD4+ T cells with immunoaugmenting properties in vivo are induced in mice within 24 h after a single injection of dimeric or aggregated IgD. In the present study, we sought to identify the region(s) of IgD responsible for upregulation of IgD-R and for the immunoaugmenting effect of IgD. IgD-R can be upregulated on CD4+ T cells in vitro and in vivo by glutaraldehyde-aggregated mutant IgD or by fragments of enzymatically digested IgD molecules possessing either the C delta 1 domain (Fd delta) or the C delta 3 domain (Fc delta). Neoglycoproteins (D-galactose--BSA and N-acetyl-D-glucosamine--BSA), can competitively block upregulation of IgD-R by IgD in vitro. Furthermore, when injected 1 day before antigen, the aggregated IgD derived molecules, KWD1 (which lacks C delta 1), KWD6 (which lacks C delta 1 plus C delta-hinge), and Fab delta can all cause augmentation of antigen-specific primary and secondary antibody responses comparable to that achieved with intact aggregated IgD. Moreover, the immunoaugmenting effect of intact oligomeric IgD molecules in primary antibody responses is competitively blocked by simultaneous injection of monomeric forms of KWD6 and Fab delta. These results suggest that the binding of IgD to IgD-R, previously shown to be dependent on N-glycans present on Fd delta and Fc delta regions, also contributes to the upregulation of IgD-R and immunoagumentation.

Characterization of membrane sugar-specific receptors in cultured high endothelial cells from mouse peripheral lymph nodes.

The culture of specialized high endothelial cells (HEC) from lymphoid organs (peripheral lymph nodes (PLN) and Peyer's patches (PP)) was undertaken in order to study and characterize the cell surface molecules which are involved in lymphocyte recognition and allow homing. Cells were stimulated in vivo by a graft versus host (GVH) type of reaction before isolation and culture. The resulting adherent and growing cells were characterized as endothelial cells because of their typical aspect and their ability to produce angiotensin-converting enzyme and factor VIII-related antigen. They possess tissue-specific endothelial addressins. MECA 79 antigen is present on cells isolated from PLN while MECA 367 antigen is detected on cells from PP. Surface receptors for glycans were studied cytochemically using neoglycoproteins and fluorescence microscopy and quantified by flow cytometry experiments which showed that the specificity of sugar receptors depends upon endothelial cell origin. Indeed, sugar receptors for alpha-L-fucosyl residues were specifically expressed by endothelial cells from PLN. These receptors were inducible upon action of activated lymphocyte-conditioned medium. Further characterization of endothelial cells from peripheral lymph nodes indicates that they indeed mediate adhesion of lymphocytes in vitro. The role of protein-sugar interactions in this process was assessed by inhibition experiments performed with the help of neoglycoproteins. Best inhibitory effects were obtained when endothelial cells had been preincubated with alpha-L-fucosyl-BSA and when lymphoid cells were preincubated with beta-D-galactosyl-BSA. Concomitant inhibition assays indicate the participation of sugar specific receptors--endogenous lectins--on the surface of both endothelial and lymphoid cells to achieve recognition and adhesion.

N-acetylcysteine and captopril protect DNA and cells against radiolysis by fast neutrons.

N-Acetylcysteine and captopril, respectively mucolytic and antihypertensive drugs, contain free sulfhydryl groups. Since in general thiols have well-established radioprotective abilities, we sought putative radioprotective effects of these drugs against therapeutic fast neutrons. We show that pBR322 plasmid DNA is indeed protected against radiolytic strand breakage by both drugs. The oxygen independent protection is consistent with a hydroxyl radical scavenging mechanism. A clonogenicity assay reveals an increase of the survival of SCL-1 cultured keratinocytes irradiated in the presence of the drugs compared with cells irradiated without drugs. Our results suggest possible interferences between treatment with drugs bearing-SH groups and radiotherapy.

A SV-40 immortalized murine endothelial cell line from peripheral lymph node high endothelium expresses a new alpha-L-fucose binding protein.

Endothelial cells from mouse peripheral lymph nodes were immortalized by cationic liposome-mediated transfection using a plasmid construct containing both the gene coding for the large T antigen of simian virus 40 and a geneticin resistance gene suitable for selection. A cell line (HECa10) was isolated on the basis of its capacity to specifically bind fucoside carrying glycoconjugates; these cells present the main characteristics of endothelial cells: production of angiotensin converting enzyme and of factor VIII-related antigen. Upon stimulation, they express E-selectin which binds oligosaccharides containing the Lewisx determinant (Fuc alpha 3[Gal beta 4 GlcNAc beta 3Gal beta) and the MECA 79 addressin which is characteristic for the peripheral lymph node high endothelium and is a L-selectin ligand. HECa10 cells, as well as peripheral lymph node high endothelial cells in primary culture, express a second fucoside binding protein which differs from E-selectin. Indeed, this new fucoside-binding protein is constitutively expressed on unstimulated cells while E-selectin is not. Furthermore, HECa10 cells mediate selective lymphoid cell adhesion in a selectin/addressin-dependent mechanism, mainly inhibited by MECA 79 antibody and, in a fucose-binding lectin-dependent manner, mainly inhibited by the specific neoglycoprotein.

C32 human melanoma cell endogenous lectins: characterization and implication in vesicle-mediated melanin transfer to keratinocytes.

To optimize skin pigmentation in order to help body prevention against UV radiation, the mechanism of melanin pigment transfer from melanocytes to keratinocytes must be elucidated. Melanin transfer to keratinocytes requires specific recognition between keratinocytes and melanocytes or melanosomes. Cell surface sugar-specific receptor (membrane lectin) expression was studied in human C32 melanoma cells, an amelanotic melanoma, by flow cytometry analysis of neoglycoprotein binding as an approach to the molecular specificity. Sugar receptors on melanocytes are mainly specific for alpha-L-fucose. Their expression is enhanced upon treatment by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which can induce melanin synthesis in amelanotic human melanoma cells in a dose-dependent manner. Flow cytometry analyses showed a small-sized population of vesicles distinguishable from large cells by their fluorescence properties upon neoglycoprotein binding. Sorting indicated that the small-sized subpopulation is composed of vesicles produced by melanocytic cells. Upon vesicle formation, a selective concentration of sugar receptors specific for 6-phospho-beta-D-galactosides appears in the resulting melanocytic vesicles. Vesicles are recognized and taken up by cultured keratinocytes and a partial inhibitory effect was obtained upon cell incubation in the presence of neoglycoproteins, indicating a possible participation of sugar receptors in this recognition. The validity for such a model to help in understanding the natural melanin transfer by melanosomes is confirmed by electron microscopy, which demonstrates the presence of melanin inside keratinocytic cells upon incubation with melanocytic vesicles.

Specificity of the murine IgD receptor on T cells is for N-linked glycans on IgD molecules.

IgD receptors on murine T cells have been reported in this issue [Tamma, S. M. L., Amin, A. R., Finkelman, F. D., Chen, Y.-W., Thorbecke, G. J. & Coico, R. F. (1991) Proc. Natl. Acad. Sci. USA 88, 9233-9237] to bind either the first or third constant region of the heavy-chain of IgD molecules--findings that could not be satisfactorily explained by IgD amino acid sequences. We now find that boiled IgD molecules or low-Mr fragments from protease-digested IgD still inhibit binding of IgD-coated erythrocytes to IgD receptors. This inhibitory activity can be absorbed with the murine IgD-binding lectin from Griffonia simplicifolia 1 (GS-1) immobilized on Sepharose. N-linked glycans, obtained from N-glycanase-treated IgD and purified by binding to GS-1-Sepharose, also inhibit rosette formation of T-helper cells bearing receptors for IgD with IgD- or mutant IgD-coated erythrocytes. Deglycosylated IgD, produced by treatment with N-glycanase, no longer binds to the lectin and fails to inhibit IgD rosetting. Binding of intact IgD to T cells is also competitively inhibited by N-acetylgalactosamine, galactose, N-acetylglucosamine, and neoglycoproteins containing these sugars. These results clearly show that N-linked glycans, present in both the first and third constant regions of the delta heavy-chain domains, are prerequisites for binding of IgD to IgD receptors.

Changes in the expression of lectins in human T lymphocyte membrane upon mitogenic stimulation.

Surface lectins, specific for given sugar structures, are expressed on human T cells, as shown by flow cytofluorometry using F-neoglycoproteins bearing either beta- and alpha-D-galactosyl, beta-D-galactosyl 6-phosphate, or alpha-L-rhamnosyl groups, but not by F-neoglycoproteins bearing other sugar groups (such as alpha-D-mannosyl groups). After stimulation with Phaseolus vulgaris mitogen, the number of cells that bind beta-D-galactosyl 6-phosphate groups (6-P-beta-D-Galp lec+ cells) increased fourfold during the first five days; these cells are helper (CD4+) T cells. Conversely, cells that bind alpha-L-Rha groups belong to the T suppressor (CD8+) family and their number moderately increased. Upon stimulation by concanavalin A, the number of cells expressing the lectin recognizing alpha-L-Rha groups increased during the first two days and then decreased within the next two days. These results are discussed with regard to the implication of lymphocyte membrane lectins in the suppressor mechanism and in the homing process.

Soluble human lymphocyte sugar binding proteins with immunosuppressive activity.

Suppression of the immune response, which involves suppressor factors released from specialized T cells, is inhibited by alpha-L-rhamnose. In this paper, we show the presence of rhamnose-specific receptors on a human CD8+ T cell-rich population and describe a novel method to isolate cells which express a given sugar-binding protein on their surface. We describe the isolation of alpha-L-rhamnose-specific molecules (rhamnose-binding fractions: RBF) from a water-soluble extract from lymphocytes, their purification by affinity chromatography on immobilized neoglycoproteins containing rhamnose residues. RBF kept their ability to bind rhamnose, as shown by the binding of fluorescein-labeled RBF to rhamnosylated BSA-substituted beads. RBF efficiently suppresses DNA synthesis of mitogen-stimulated human lymphocytes as well as B cell immunoglobulin production. Therefore, these rhamnose-binding molecules appear to be antigen-independent suppressor factors.