Effects of <i>Blastocystis</i> lysate on LPS stimulation of THP1-Blue monocytes.

<p>A. Detection of SEAP activity from cell culture supernatants of THP1-Blue monocytes incubated with various conditions. <i>Blastocystis</i> total cell lysate incubated with THP1-Blue cells at amounts corresponding to ratio of 1 monocyte to 10 parasites. <i>Blastocystis</i> ST7-B, not ST4-WR1, significantly augments LPS-induced activation of THP1-Blue monocytes, as observed in the increased SEAP activity. B. RT-qPCR data of SEAP gene in THP1-Blue cells. Significant increase in fold change of SEAP gene expression observed only in THP1-Blue cells co-cultured with ST7-B WCL and LPS. C. Heat-inactivated lysate of both ST7-B and ST4-WR1 shows augmenting effect on LPS-induced THP1-Blue activation. **, <i>p</i><0.01; *, <i>p</i><0.05, when analyzed against 1 µg/ml LPS positive control.</p

Effects of live <i>Blastocystis</i> on LPS stimulation of THP1-Blue monocytes.

<p>A. Representative images of colorimetric changes to THP1-Blue cell culture supernatant, indicating SEAP activity of substrate medium. B. Inter-isolate variation in live <i>Blastocystis</i> in modulating effects on LPS stimulation of THP1-Blue monocytes. Detection of SEAP activity from cell culture supernatants of THP1-Blue monocytes incubated with various conditions. Live parasites incubated with THP1-Blue cells in numbers corresponding to ratio of 1 THP1-Blue cell to 10 parasites (1–10), or 1 THP1-Blue cell to 20 parasites (1–20). Live ST4-WR1 and not ST7-B significantly dampens LPS-mediated NF-κB activation in THP1-Blue monocytes. Live parasites of both isolates also significantly reduce background absorbance. C. AnnexinV-FITC PI viability assessment of THP1-Blue monocytes after exposure to live parasites. Slight but consistent decrease in viability in THP1-Blue monocytes after exposure to live parasites of both isolates. **, <i>p</i><0.01, when analyzed against 1 µg/ml LPS positive control. ##, <i>p</i><0.01, when analyzed against complete media negative control.</p

Effect of NaHS and capsazepine on protein and mRNA levels of SP in septic mice.

<p>Mice were randomly given NaHS (10 mg/kg, i.p.) or vehicle (DMSO) at the same time of CLP; and capsazepine (<i>Capz</i>) (15 mg/kg, s.c.) or vehicle (DMSO) 30 minutes before CLP. Sham mice were used as controls. Eight hours after CLP or sham operation, (A) lung and (B) plasma SP levels, and (C) lung PPT-A mRNA levels were measured. Results shown are the mean values ± SEM (n = 8–12 mice per group). *P<0.01 versus sham; **P<0.01 versus CLP+vehicle; ‡P<0.05 versus CLP+vehicle; †P<0.01 versus CLP+NaHS+vehicle.</p

<i>Blastocystis</i> effects on zymosan (ZG) stimulation of THP1-Blue monocytes.

<p>Detection of SEAP activity from cell culture supernatants of THP1-Blue monocytes incubated with various conditions. A, B. Both <i>Blastocystis</i> ST7-B and ST4-WR1 significantly inhibits ZG-induced activation of THP1-Blue monocytes, as observed in the decrease in SEAP activity. Parasite lysate was added to THP1-Blue cells in terms of amounts of lysate prepared from 1 million (mil), 5 million, or 10 million parasites per ml. Inhibition observed to be in negative correlation with parasite lysate concentration, with significance observed from 5×10⁶ ST7-B/ml onward. **, <i>p</i><0.01, when analyzed against 5 µg/ml ZG positive control.</p

<i>Blastocystis</i> effects on flagellin (fla) stimulation of THP1-Blue monocytes.

<p>Detection of SEAP activity from cell culture supernatants of THP1-Blue monocytes incubated with various conditions. Parasite lysate was added to THP1-Blue cells at amounts that correspond to a ratio of 1 THP1-Blue cell to 10 parasites. Both <i>Blastocystis</i> ST7-B and ST4-WR1 does not significantly modulate fla-induced activation of THP1-Blue monocytes.</p

Schematic summary of signaling events in H₂S-induced neurogenic inflammation in a murine model of polymicrobial sepsis.

<p>H₂S has been demonstrated to be overproduced in sepsis. H₂S stimulation of TRPV1 and the downstream release of SP lead to the activation of ERK_1/2, which subsequently induces the phosphorylation and degradation of IκBα, as well as the translocation and activation of NF-κB, thereby leading to SIRS and MODS characteristic of severe sepsis. indicates exogenous administration; indicates inhibition.</p

Effect of PAG and capsazepine on liver dysfunction and lung edema in septic mice.

<p>Mice were randomly given PAG (50 mg/kg, i.p.) 1 hour before (“prophylactic”) or 1 hour after (“therapeutic”) CLP; and capsazepine (<i>Capz</i>) (15 mg/kg, s.c.) or vehicle (DMSO) 30 minutes before CLP. Sham mice were used as controls. Eight hours after CLP or sham operation, plasma levels of (A) ALT and (B) AST, and (C) lung wet-to-dry weight ratio were measured. Results shown are the mean values ± SEM (n = 10–15 mice per group). *P<0.01 versus sham; **P<0.01 versus CLP+vehicle; ‡P<0.05 versus CLP+vehicle.</p

Effect of NaHS and capsazepine on liver dysfunction and lung edema in septic mice.

<p>Mice were randomly given NaHS (10 mg/kg, i.p.) or vehicle (DMSO) at the same time of CLP; and capsazepine (<i>Capz</i>) (15 mg/kg, s.c.) or vehicle (DMSO) 30 minutes before CLP. Sham mice were used as controls. Eight hours after CLP or sham operation, plasma levels of (A) ALT and (B) AST, and (C) lung wet-to-dry weight ratio were measured. Results shown are the mean values ± SEM (n = 10–15 mice per group). *P<0.01 versus sham; **P<0.01 versus CLP+vehicle; ‡P<0.05 versus CLP+vehicle; †P<0.01 versus CLP+NaHS+vehicle.</p

Effect of NaHS or PAG and capsazepine on ERK_1/2 activation in the lungs and liver of septic mice.

<p>Mice were randomly given NaHS (10 mg/kg, i.p.) at the same time of CLP or PAG (50 mg/kg, i.p.) 1 hour before (“prophylactic”) or 1 hour after (“therapeutic”) CLP; and capsazepine (<i>Capz</i>) (15 mg/kg, s.c.) or vehicle (DMSO) 30 minutes before CLP. Sham mice were used as controls. Eight hours after CLP or sham operation, effect of (A) NaHS or (B) PAG and capsazepine on phospho-ERK_1/2 and total ERK_1/2 expression levels were measured. A representative western blot image is shown, with densitometry data expressed as average ratios of phospho-ERK_1/2 to total ERK_1/2. Results shown are the mean values ± SEM (n = 6 mice per group). *P<0.01 versus sham; **P<0.01 versus CLP+vehicle; †P<0.01 versus CLP+NaHS+vehicle.</p

Effect of PAG and capsazepine on protein and mRNA levels of SP in septic mice.

<p>Mice were randomly given PAG (50 mg/kg, i.p.) 1 hour before (“prophylactic”) or 1 hour after (“therapeutic”) CLP; and capsazepine (<i>Capz</i>) (15 mg/kg, s.c.) or vehicle (DMSO) 30 minutes before CLP. Sham mice were used as controls. Eight hours after CLP or sham operation, (A) lung and (B) plasma SP levels, and (C) lung PPT-A mRNA levels were measured. Results shown are the mean values ± SEM (n = 8–12 mice per group). *P<0.01 versus sham; **P<0.01 versus CLP+vehicle.</p