caseolyticus and 99±1% (97.3±1.5% at 24 h) – untreated cells. Thus, there are no apparent or systematic differences in macrophage viability during the initial 6-h incubation period of the experiment, corresponding to the period of cytokine peak. In light

Ruxolitinib concentration of the in vitro proinflammatory cytokine induction of S. iniae EPS, we were next interested in determining whether similar events also occur in vivo, and in revealing the clinical outcomes following EPS inoculation. To accomplish this we first constructed a dose-effect (lethal) model. Mortality rates were affected by both time and group (EPS/LPS dosages). As shown in Fig. 3, EPS induced death of fish in a dose-dependent fashion: low doses (0.55 mg per fish) resulted in 10% mortality, while higher doses resulted in increased

mortality rates (P<0.001). Administration of 2.2 mg of EPS per fish resulted in 60% mortality within the first 24 h, while 1.1 mg of EPS per fish yielded 40% mortality during the same period (P<0.01 between these doses). Mortality in fish injected with the higher doses continued for several more days, cumulating in 90% at 144-h postinoculation, resembling that of the LPS-induced septic shock in a mouse model (An et al., 2008) and the (24-h delayed) LPS-induced mortality (80%) of trout observed in the present work (Fig. 3). None of the PBS-injected fish succumbed. anti-CTLA-4 antibody Gross pathological findings in dead and moribund fish consisted in discoloration of skin (mainly around the tail), presence of ascitic fluids in the celomic cavity and inflammation with ecchymotic hemorrhages in the gut and peritoneum. Thus, 1.1 mg of EPS per fish was used as the effective dosage in subsequent experiments where cytokine-specific mRNA transcripts levels were assessed. Relative cytokine mRNA levels analysis revealed that augmentation of specific transcripts was significantly superior to that of the in vitro system. Following inoculation

of EPS, TNF-α2 Florfenicol transcription levels peaked at 12-h postinjection (1320-fold increase) and remained elevated for a considerable time (71-fold increase at 24 h), whereas TNF-α1 transcription levels, peaking at 12-h postinjection, were relatively lower (18.1-fold increase) and decreased to a 2.8-fold increase at 24 h (P<0.01 for the difference between the two cytokines) (Fig. 4). LPS injection (Fig. 5) resulted in 115.4-fold increase of TNF-α2 transcripts (remaining elevated throughout the experiment) and 25.9-fold increase of TNF-α1 transcripts (at 9 h). Differences between the two cytokines were nonsignificant. Injection of PBS (negative control) did not affect cytokine transcription levels. IL-1 transcript level among the EPS-injected fish was increased by 209-fold; IL-6 transcript level of the same fish was increased 560.9-fold. LPS-injected fish showed a 252.1-fold increase of IL-1 transcripts and a 536.7-fold increase of IL-6 transcripts (P<0.001). All of the IL transcripts peaked at 6–9-h postinjection.