Measurement of the impedance of the HBEC monolayers during coculture with IRBC.

Opening of the intercellular junctions was estimated by dynamic measurement of the impedance of the monolayer every 10 min for 24 h after the beginning of the co-culture on an ECIS instrument. HBEC monolayers are grown to confluence and co-cultured with IRBC (3Ci- or CS2) or NRBC. Red blood cells were removed from the HBECs by gentle wash, 4 hours after beginning of incubation. All the monolayers are incubated with 10 ng/ml of TNF prior commencement of the co-culture with no effect on the impedance (see Controls). Experiments were done more than 10 times but only illustrative curves are shown.. Nocodazole (10 µM), rolipram (10 µM) or anti-ICAM-1 (5 µg/ml) were added 30min prior beginning of the co-culture and let in the medium during all the time. TNF (100 ng/ml) and Histamine (100 µM) were used as positive control to induce opening of the junction. A) show decrease of impedance when HBEC monolayer is co-cultured with IRBC(3Ci) or IRBC(3Ci)+NRBC (vol/vol), but not when co-cultured with normal RBC). Impedance is related to parasiteamia as the effect is less when IRBC are diluted with NRBC; B) decrease of impedance when HBEC monolayer is co-cultured with IRBC(3Ci) but not with IRBC(CS2) which do not bind tightly to HBECs; C) inhibition of the IRBC(3Ci) effect on impedance by previous incubation of the monolayer with nocodazole; D) absence of inhibitory effect of rolipram on impedance in the same setting as C); E) absence of inhibitory effect of anti-ICAM1 on impedance in the same setting as C).