Degraded CGN Induced-TNF-α Production by Monocytes In Vitro 
In order to study the capacity of dCGN to stimulate TNF-α production, peripheral blood monocytes were cultivated in the presence of dCGN (0.1 to 1 mg/ml). Very low levels of TNF-α were induced in PBM after stimulation with native CGN (Fig. 2A). Addition of 0.1 mg/ml 10 kDa dCGN resulted in approximately a 60-fold increase in TNF-α production by PBM. This was a dose-dependent effect that reached a 180-fold increase when cells were exposed to 1 mg/ml of 10 kDa dCGN (Fig. 2A). A 250-fold increase in TNF-α production was detected at 1 mg/ml 40 kDa dCGN (Fig. 2A). TNF-α production increased in time reaching a maximum level at 8 hours of culture (Figure 2B). After 24 h, the amount of secreted TNF-α was still one third of the total TNF-α. Lipopolysaccharide (LPS), a known activator of immune cells also induced TNF-α production with similar kinetics as dCGN (Fig. 2B). However, the amount of TNF-α produced by LPS was 4-fold less than the one produced by dCGN and it was not detected after 8 hours of culture (Fig. 2B). Similarly, monocytic THP-1 cells cultivated in the presence of variable concentration of dCGN showed an increase in TNF-α production (Fig. 2C). This increase in TNF-α production was significantly smaller (about 10-fold) than the one presented by PBM (Fig. 2A). No TNF-α was released from THP-1 cells exposed to native CGN (not shown). TNF-α production by THP-1 cells was not dose dependent to the amount of dCGN used. Also there was no difference between the two forms (10 and 40 kDa) of dCGN (Fig. 2C). Interestingly, TNF-α release from THP-1 cells stimulated with dCGN reached a maximum level at 32 h, while stimulation with LPS reached a maximum level at 56 h (Fig. 2D).
10.1371/journal.pone.0008666.g002 Figure 2  Degraded CGN stimulated TNF secretion from monocytes.
Levels of TNF released from peripheral blood monocytes (A–B) and THP-1 cells (C–D) after stimulation with dCGN. A: TNF release induced by native CGN (open bars), 10 kDa dCGN (grey bars), or 40 kDa dCGN (black bars). B: Kinetics of TNF release induced by nothing (control; black diamonds), 0.1 mg/ml 10 kDa dCGN (black triangles), 0.1 mg/ml 40 kDa dCGN (black squares), or 10 µg/ml LPS (open squares). C: TNF release induced by nothing (control; open bars), 10 µg/ml LPS (hatched bars), or increasing concentrations of 10 kDa dCGN (grey bars), or 40 kDa dCGN (black bars). D: Kinetics of TNF release induced by nothing (control; black diamonds), 0.1 mg/ml 10 kDa dCGN (black triangles), 0.1 mg/ml 40 kDa dCGN (black squares), or 10 µg/ml LPS (open squares).

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