However, when LMP-420 was added 2 h before or simultaneously with TNF or LT, up-regulations observed for ICAM-1 and/or VCAM-1 with both stimuli were dramatically inhibited (Dunn’s test, 0.01 in each condition), and the weak induction of CD40 by the two cytokines was abrogated. Open in a separate window Figure 1 Structure of LMP-420 (2-NH2-6-Cl-9-[(5-Dihydroxyboryl)-Pentyl] Purine) Open in a separate window Figure 2 Inhibitory Effect of LMP-420 on TNF-Induced Up-Regulation of KRAS2 ICAM-1 and VCAM-1HBEC-5i were left unstimulated or were activated with LMP-420 alone, TNF alone, TNF with LMP-420, LT alone, or LT with LMP-420. tumor necrosis factor (TNF) in the pathogenesis of CM, and a clear relationship has been established between plasma concentrations of TNF levels and cerebral pathology [6C8]. In experimental CM, TNF-beta, now called lymphotoxin (LT), was recently shown to be the principal mediator of pathogenesis [9]. Indeed, LT and TNF belong to the same RN-1 2HCl family, interact with a common receptor, and could act together during the pathogenesis [10]. Both cytokines can activate endothelium and be responsible for an increase of MP release by human brain endothelium (S. C. Wassmer, V. Combes, F. Candal, I. Juhan-Vague, and G. E. Grau, unpublished data) [11]. In this report we test the anti-inflammatory activity of a newly designed 2-NH2-6-Cl-9-[(5-dihydroxyboryl)-pentyl] purine, named LMP-420. LMP-420 inhibits transcription of mRNA RN-1 2HCl for TNF in a variety of human cell types including monocytes, T lymphocytes, neutrophils, adipocytes, and endothelial cells (ECs), and has a 50% inhibitory concentration (IC50) of 50 nM in human peripheral blood mononuclear cells (S. Haraguchi, N. K. Day, W. Kamchaisatian, M. Engele, S. Stenger, N. Tangsinmankong, J. W. Sleasman, S. V. Pizzo, and G. J. Cianciolo, unpublished data). In this study, using an in vitro co-culture model composed of human brain microvascular EC (HBEC-5i), and FCR-3 or FCR-3Cderived strains, we aimed to assess the ability of LMP-420 to inhibit in vitro TNF and/or LT effects on brain endothelium, with particular attention to its activation, adhesiveness for malarial parasites, and vesiculation. Methods Reagents LMP-420 (2-NH2-6-Cl-9-[(5-dihydroxyboryl)-pentyl] purine) was provided as a gift from LeukoMed, Inc. of Raleigh, North Carolina, United States. It was stored either as a dry powder under desiccation at ?20 C or at ?20 C as aliquots of a 10 mM stock solution in DMSO (tissue culture grade dimethylsulfoxide; Sigma, St. Louis, Missouri, United States). Human Brain Endothelial Cells (HBEC-5i) Purified human brain microvascular EC (HBEC-5i [12]) were seeded on culture flasks and produced to confluence in DME/F12 medium (pH 7.4) supplemented with 10% fetal bovine serum, 30 g/ml endothelial cell growth supplement, and 10 g/ml gentamycin. Parasites FCR3, RP8 (able to bind CSA), and PAC2 (able to bind CD36 and ICAM-1) parasites were cultured on human 0+ erythrocytes in candle jars as described [13]. They were produced under standard culture conditions, replacing the 10% v/v human serum with 0.25% w/v Albumax (Life Technology, Paris, France). PRBC preparations were enriched to 80%C85% by gelatin flotation with Plasmion (Fresenius Kabi France, Couvier, France) [14], and suspensions were adjusted to 5 106 PRBC/ml for cytoadherence assays. Inhibition of HBEC ICAM-1 and VCAM-1 Up-Regulation by LMP-420 upon TNF and LT Activation HBEC-5i confluent monolayers were left unstimulated with and without treatment with LMP-420 (50 nM), or were activated with TNF (overnight or 6 h, 10 ng/ml) or with LT (overnight, 30 ng/ml), concomitantly or not with LMP-420 (50 RN-1 2HCl nM), before analysis. HBEC-5i were then harvested and labeled by indirect labeling using mouse anti-human CD54 (ICAM-1 [84H10]) and CD106 (VCAM-1 [1G1]) antibodies (Beckman-Coulter Immunotech, Marseille, France), CD40 monoclonal antibody (mAb) (B-B20, Diaclone, Besan?on, France), and CD36 mAb (FA6C152, gift from L. Edelman, Institut Pasteur Paris) as the first step. Secondary goat anti-mouse Alexa488Ccoupled mAb (Molecular Probes, Eugene, Oregon, United States) was added as the second step. A nonspecific isotype-matched mouse IgG1 (Beckman-Coulter Immunotech) was used for all controls. Cells were then resuspended in PBS before flow cytometry analysis on a Coulter Epics XL (Coultronics France, Margency, France). The area corresponding to HBEC-5i was defined, and mean fluorescence intensities of the positive cell populations were measured for each antigen. Effect of LMP-420 on Several Parasite Strains Cytoadherence to Activated HBEC For cytoadherence assays, HBEC-5i were plated RN-1 2HCl on 1% w/v gelatin-coated 12-well IFA slides and allowed to reach.