Indeed, as showed by TUNEL assay, exposure of TEC selleck bio for 48 hours to septic plasma induced a significant increase of apoptosis in respect to healthy plasma (Figure (Figure3a).3a). However, when TEC were cultured for 48 hours in the presence of Amberchrom resin-adsorbed plasma, the apoptotic rate was significantly reduced (Figure (Figure3a).3a). The inhibition of plasma-induced apoptosis was observed after incubation of TEC with samples obtained after 15, 30, 60 and 120 minutes from the beginning of adsorption. The maximal inhibition of plasma-induced apoptosis of TEC was detected with samples obtained after 120 minute adsorption (Figure (Figure3a).3a). LPS (30 ng/ml) was used as a positive control (Figure (Figure3a).3a).

Interestingly, the addition in culture of 5 ��g/ml polymyxin B significantly reduced but did not completely abolish the pro-apoptotic activity of septic plasma (Figure (Figure3a).3a). These results were confirmed by counting nuclear fragmentation, a typical feature of apoptotic cells, after propidium iodide staining (not shown). Moreover, the pre-incubation with septic plasma induced a significant increase of TEC apoptosis in the presence of LPS and inflammatory cytokines (Figure (Figure3b).3b). This effect was not observed with plasma previously subjected to resin adsorption or with healthy plasma (Figure (Figure3b).3b). In accordance to the TUNEL data, the activities of caspases-3, -8 and -9 were significantly increased in TEC incubated with septic plasma. In contrast, a significant reduction of all caspase activities was observed in TEC cultured in the presence of Amberchrom resin-treated plasma (120 minutes of treatment; Figure Figure3c).

3c). These results suggest that plasma-induced TEC apoptosis was predominantly associated to the activation of the death-receptor pathway induced by soluble mediators. Indeed, the knock-down of TNF-R1, Fas and CD40 in TEC by specific siRNA significantly decreased the pro-apoptotic activity of septic plasma (Figure (Figure4a).4a). We also found that supernatants collected from CHO cells transfected with human Fas-L cDNA induced a significant increase of septic plasma-associated apoptosis (Figure (Figure4b).4b). The apoptotic rate of plasma-treated TEC was not affected by supernatants derived from mock-transfected CHO cells (Figure (Figure4b).4b).

These data suggest that septic plasma induced a sensitization of TEC to Fas-mediated apoptosis. Amberchrom resin adsorption abrogated the sensitization of TEC to Fas-mediated apoptosis (Figure (Figure4b).4b). The sensitization of TEC to Fas-mediated apoptosis may be ascribed to the up-regulation of Fas on TEC surface induced by septic plasma that was not Brefeldin_A observed after Amberchrom resin adsorption (Figures (Figures4c4c and and4d).4d). In addition, Amberchrom resin adsorption reduced the concentration of pro-apoptotic soluble plasma factors.