1 Introduction Plasmodium falciparum Brown et al., 1999a,b, 2001a Wassmer et al., 2004; Combes et al., 2006 Combes et al., 2005, 2006 One question is the mechanism underpinning the accumulation of PRBC leading to micro-vascular occlusion as seen in post mortem cerebral malaria (CM) brain tissue, which is believed to be a progressive phenomenon of PRBC sequestration. We suggest that the initial sequestration of PRBC, if maintained for a prolonged period of time, has the ability to activate the endothelium to promote sequestration, leading to deleterious effects on the host. Our studies investigate the direct effects on the endothelium of PRBC retention for prolonged periods of time. low Shiu and McIntire, 2003 Brown et al., 2001b; Shiu and McIntire, 2003; Viebig et al., 2005; Tripathi et al., 2006 Pino et al., 2003 Shiu et al., 2002 Pino et al., 2003 Viebig et al., 2005 Lyke et al., 2004 Burgmann et al., 1995 Hermsen et al., 2003 Wolff et al., 1998; Utgaard et al., 1998; Oynebraten et al., 2004 Shiu et al., 2000 Bisser et al., 2006 Lucas et al. (1997a) Lucas et al., 1997b P. falciparum Wenisch et al., 1994 Lou et al., 1998 Lucas et al., 1997a http://www.affymetrix.com Our results have led us to propose a novel mechanism for the modulation of the endothelium during malaria infection that is dependent on low level TNF and involves a pro-inflammatory component but also a concurrent down-modulation of RBC-induced inflammation due to the presence of the parasite within the infected cell. 2 Materials and methods 2.1 Malarial parasites Plasmodium falciparum Ockenhouse et al., 1992 Gray et al., 2003 l N N Trager and Jensen, 1976 2.2 Endothelial cells Pooled human umbilical vein endothelial cells (HUVEC) were obtained from Promocell (Heidelberg, Germany), HUVEC from different batches were used for each experiment, at passages three to five. HUVEC were grown to confluence on 1% gelatin (Sigma, UK) coated flasks and plates. All co-culture experiments were performed in serum-depleted basal HUVEC medium (quiescing medium) which consisted of M199 (Invitrogen, UK) containing 1% FCS. These conditions were designed to increase the signal window, while maintaining the integrity of the HUVEC monolayer (i.e. there was no indication of cell apoptosis or necrosis during the experiments). low high 2.3 PRBC-EC co-culture conditions 2 low 2.4 RNA expression: microarray analysis Following incubation, the supernatant was aspirated, HUVEC was washed with cold RPMI-1640 and then with 0.02 M EDTA to remove the adherent RBCs and the cells harvested using Trizol (Invitrogen, UK). RNA integrity was evaluated by electrophoresis on a 1% agarose gel and by spectrophotometry using the absorbance ratio at 260/280 nm. low low low high Gentleman et al., 2004 Irizarry et al., 2003 http://www.r-project.org/ Gautier et al., 2004 P Hochberg and Benjamini, 1990 B F P http://www.geneontology.org http://bioconductor.org/packages/1.9/bioc/html/GOstats.html P P 2.5 ICAM-1 protein expression low g The HUVECs were washed once with cold RPMI-1640 and then with 0.02 M EDTA to remove the adherent RBCs and subsequently harvested by trypsinisation for analysis by flow cytometry. FACS ICAM-1 expression on HUVEC was determined by staining the cells using a fluorescein isothiocyanate (FITC)-conjugated mouse anti-human ICAM-1 antibody (MCA1615F; Serotec) using standard staining protocols and the cells fixed in 2% paraformaldehyde and analysed by flow cytometry. ICAM-1 expression was expressed as geometric mean of the fluorescence intensity. 2.6 IL-8 and TNF receptor expression The supernatants stored from the co-culture studies were analysed using a standard sandwich ELISA kit (IDS), using a horse-radish peroxidise based colorimetric detection system, to quantify IL8 released from ECs. IL8 production was expressed as a concentration in pg per ml. Similarly, soluble TNFR I, sTNFR I (p55) and soluble TNFR II, sTNFR II (p75), were detected using sTNFR I (KAC1761) and sTNFR II (KAC1771) ELISA kits (Biosource). TNFR level was expressed as TNFR concentration in ng per ml. In order to understand the kinetics of TNFR expression on the surface of ECs in response to co-culture with PRBCs, the ECs were co-cultured with PRBCs and uninfected RBCs for 0.5, 1, 2 and 3 h. Following the incubation period, HUVECs were harvested and dual stained for surface TNFRs with monoclonal anti-human RII-FITC (FAB226F) and monoclonal anti-human RI-PE (FAB226F) antibodies (R&D Systems Europe). The receptor expression was expressed as the geometric mean of the fluorescence intensity. 2.7 Trypsin digestion of RBC Chaiyaroj et al., 1994 Gray et al., 2003 2.8 Transwell experiments Confluent HUVECs were co-cultured with PRBCs separated using a 0.4 μm transwell filter (Falcon), which prevents contact between HUVECs and RBCs but allows soluble factors to diffuse through. This set-up allowed us to determine whether contact between RBCs and ECs was necessary for induction of ICAM-1 expression on the ECs. 3 Results 3.1 Up-regulation of ICAM-1 expression following co-culture with infected and uninfected RBC Fig. 1 low P low Fig. 1 3.2 Up-regulation of IL-8 release following co-culture with infected and uninfected RBC P Fig. 2 low P low P Fig. 2 3.3 Up-regulation of soluble TNF receptor release and down-regulation of surface expression following co-culture with infected and uninfected RBC P Fig. 3 P P Fig. 4 3.4 Loss of ICAM-1-mediated adhesion phenotype following trypsinisation of PRBC Fig. 5 low Fig. 5 3.5 Modulation of the endothelial cell transcriptome following co-culture with infected and uninfected RBC Fig. 6 Fig. 6 low low Fig. 6 P low Fig. 6 low low low low Fig. 6 low low Fig. 6 Nanami et al., 2005 Basilico et al., 2004 Fig. 7 Supplementary Fig. S1(see on-line supplementary data) Supplementary Tables 1, 2 and 3; see on-line supplementary data P low low Table 1 Table 2 low P low low Table 2 Schwartz et al., 1994 Gijsbers et al., 2005 low low low Table 2 Hatabu et al., 2003 Table 2 low Table 2 low Table 2 Table 3 Ho and Hawkins, 2005 low Utz and Anderson, 2000; Perrin and Huttenlocher, 2002 low 4 Discussion low low Randolph and Furie, 1996; Pino et al., 2003; Viebig et al., 2005 Viebig et al. (2005) Pino et al. (2003) Tripathi et al., 2006 Wautier et al., 1983 Brown et al., 2001b Walmet et al., 2003 Eyler and Telen, 2006 Wautier and Schmidt, 2004 Hermand et al., 2003 Wautier and Wautier, 2004 Aderka et al., 1998 P. falciparum Wenisch et al., 1994 Lukacs et al., 1995 low Fig. 6 low Fig. 6 low Brown et al., 2001b Brown et al., 2001b; Wautier et al., 2001 Wautier et al., 2001 Lucchi et al., 2006 Yipp et al., 2003 Brown et al., 1999a Medana and Turner, 2006 Supplementary data doi:10.1016/j.ijpara.2007.02.006 Supplementary data Supplementary data