05). Although relatively low, coculture of RTS11 macrophages with live or killed cells of the commensal S. caseolyticus KFP 776 still induced an increase in transcription levels (2.6±0.5–3.0±0.6-fold increase). But, in contrast to the early rise of proinflammatory cytokine transcriptional levels that was typical of true pathogens, the commensal strain induced only a late and minor increase. For IL-6, LPS and live S. iniae-induced augmented mRNA transcription levels that peaked at an earlier time (6-h poststimulation) compared with heat-killed (A. salmonicida or S. iniae) or live A. salmonicida bacteria (9-h poststimulation). As was the case for TNF-α and IL-1, stimulation with commensal S. caseolyticus induced only

a Oligomycin A concentration small (and late, for killed cells) increase in levels of mRNA transcription levels (Fig. 1). To further delineate the role of S. iniae EPS in the induction of cytokine transcriptions, RTS-11 macrophages were stimulated by purified EPS. The experimental set was analogous to other in vitro experiments and was performed contemporaneously with those where whole bacterial cells were used. Figure 2, illustrating the magnitude and the kinetics of TNF-α, IL-1 and IL-6

transcriptional levels induced by equivalent concentrations of EPS or LPS during 24 h of incubation, indicates that although both substances are highly effective in inducing an early response Apoptosis inhibitor (6–9-h poststimulation), EPS is more efficient than LPS in all parameters tested. In terms of TNF-α mRNA transcript induction, S. iniae EPS induced significant activation of macrophage, which resulted in mRNA transcription levels that are practically double those observed after LPS stimulation [5.2±0.8- vs. 10.4±1.4-fold for TNF-α1 (P<0.005), and 5.7±0.6- vs. 10±1.5-fold increase for TNF-α2 (P<0.01)]. Similar results were detected with IL-1 transcription levels [5.3±1.7-fold increase with LPS; 10.3±1.3-fold increase with EPS (P<0.05)]. The highest degree of transcription levels was that of IL-6 (43±7.9-fold increase with EPS; 8.8±2.3-fold increase with LPS). The plausibility that the observed Etofibrate differences in levels of cytokine transcription are related to macrophage viability was

assessed by determining cellular ATP levels (Mossmann, 1983). When macrophages were stimulated (by LPS or EPS), infected by live S. iniae or A. salmonicida or cocultured with killed S. iniae or A. salmonicida, the percentages of living macrophages at 6-h poststimulation, as determined in quadruplicate experiments, were as follows: 97.3±1.5% (80.7±1.5% at 24 h) – EPS-stimulated cells; 97.3±1.5% (70±2.6% at 24 h) – LPS-stimulated cells; 90±2% (75±2% at 24 h) – coculture with killed A. salmonicida; 75±2% (19.7±3.5% at 24 h) – infection with live A. salmonicida; 98.3±1.5% (74±2.6% at 24 h) – coculture with killed S. iniae; 97.3±1.5% (50.7±3.1% at 24 h) – infection with live S. iniae; 97.7±1.5% (91.7±3.5% at 24 h) – coculture with killed S. caseolyticus commensal strain; 90.7±2.1% (48.7±4.