Effects of blockade of PAF receptors by intrathecal injection of PAF receptor antagonists (A–D) or knockdown of spinal PAF receptors by siRNA (E–H) on tactile allodynia (A, B, E, F), guarding behavior (C, G) and limb-use abnormality (D, H) in FBC mice.

<p>(A–D); Tumor cells were implanted into the intramedulla of left femur bone 13 days before the intrathecal (i.t.) injection of PAF receptor antagonists. TCV-309 (10 pg/mouse), WEB 2086 (10 pg/mouse) or the vehicle were injected intrathecally at time “0”. Data are expressed as the mean ± SEM., n = 8–12 mice per group. *P<0.01 compared with the corresponding control (vehicle treated) values, as determined by analysis of variance followed by Tukey-Kramer test. Control mice received injections with a vehicle: ACSF. The pain-related behaviors that developed after tumor implantation in mice were not affected by the vehicle treatments. (E–H); siRNA or mismatched siRNA of PAF-receptor mRNA were transfected into the spinal cord 13 days after tumor implantation. Data are expressed as the mean ± SEM. n = 8–10 mice per group. †P<0.05, *P<0.01 compared with the corresponding control (mismatched siRNA transfection) values, as determined by analysis of valiance followed by an unpaired Student's <i>t</i>-test. Control mice received injections with mismatched siRNA or a vehicle: HVJ-envelope only. The pain-related behaviors that developed after tumor implantation in mice were not affected by mismatched siRNA or vehicle treatments.</p

Enhanced pain reliving effect with a combination of TCV-309 and morphine in FBC mice.

<p>TCV-309, 3 and 10 μg/kg were injected i.v. at 10 days post tumor implantation and morphine 0.1 mg/kg and 0.3 mg/kg were injected s.c. 1 day after the injection of TCV-309 (A). Gabapentine 10 and 30 mg/kg i.v. were injected 11 days post tumor transplantation and morphine, 1 mg/kg was injected at 30 min after gabapentin injection (B). Morphine 0.3–30 mg/kg was injected at 11days post tumor transplantation (C). Pain-like behaviors were evaluated at 20 min after morphine injection. Various doses of TCV-309 were injected i.v. at 11 days post tumor implantation and morphine 0.3 mg/kg were injected s.c. 1 days after the injection of TCV-309 (D). One day after the injection of TCV-309, various doses of morphine were injected (E). Control mice received injections with a vehicle: saline. The pain-related behaviors that developed after tumor implantation in mice were not affected by vehicle treatments. Each group contained 11 mice. ^†P<0.05, *P<0.01 compared with the corresponding control (vehicle treated) values, as determined by analysis of variance followed by Tukey-Kramer test. ^§P<0.01 compared with the corresponding control (vehicle treated without morphine) values, as determined by analysis of variance followed by an unpaired Student's <i>t</i>-test.</p

Enhanced pain reliving effect of TCV-309, WEB 2086, BN 50739 and morphine in FBC mice.

<p>TCV-309, WEB 2086 and BN 50739 were administered at 11 days post tumor implantation. Morphine 0.3 mg/kg s.c. was injected at 8 days after the injection of PAF receptor antagonists. Allodynia (A, B), guarding behavior (C) and limb-use abnormality (D) were evaluated at 20 min after the injection of morphine. Values represent the mean ± SEM. n = 11 mice per group. *P<0.01 compared with the corresponding control values, as determined by analysis of variance followed by an unpaired Student's <i>t</i>-test.</p

Effect of the repeated administration of TCV-309 on the pain-like behaviors in FBC mice.

<p>The administration of TCV-309 0.3 mg/kg i.v. was started 6 hr before the tumor implantation, given once a day and continued every 4 days up to 28 days. Allodynia (A, B), guarding behavior (C) and limb-use abnormality (D) were evaluated at 3 hr and 1, 2, 3 days after TCV-309 injection. Data are expressed as the mean ± SEM. n = 15 mice per group.</p

The Kaplan-Mayer survival curve of FBC mice and the change of body weight (insert).

<p>For the survival experiments, TCV-309 and saline were given once a day and continued every 4 days until the animals died (n = 17 and 50, respectively). Control mice received saline for 32 days. Days for 50% of mice died after receiving TCV-309 were significantly prolonged compared to the saline-treated control, P<0.001. Statistical analysis was performed by log-rank and Gehan–Breslow–Wilcoxon tests.</p

Changes in lysophosphatidylcholinr acyltransferase 2 (LPCAT2), an inducible PAF synthesis enzyume, in the spinal cords of mice after implantation of tumor cells.

<p>Alteration of spinal LPCAT2 expression 3, 8, 15, and 30 days after the levels of immunoreactivity were normalized to that of β-actin and represented as % induction compared with the values of sham-operated (deaden cells implanted) mice (mean ± SEM., n = 5–7). *P<0.05, ***P<0.001 versus the corresponding values in sham-operated mice (unpaired Student's <i>t</i>-test).</p

Morphine-induced constipation with or without TCV-309.

<p>Normal mice received with various doses of morphine and the accumulated feces on the floor over 60-309 was injected 1 day before morphine administration. Data are expressed as the mean. n = 10 mice per group.</p

Phospholipase C-Related Catalytically Inactive Protein Participates in the Autophagic Elimination of <i>Staphylococcus aureus</i> Infecting Mouse Embryonic Fibroblasts

<div><p>Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP) that was originally identified as an inositol trisphosphate-binding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of <i>Staphylococcus aureus</i> in infected mouse embryonic fibroblasts (MEFs). We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of <i>S. aureus</i> cells in <i>PRIP</i>-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that <i>S. aureus</i> proliferates in LC3-positive autophagosome-like vacuoles in <i>PRIP</i>-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in <i>PRIP</i>-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in <i>PRIP</i>-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of <i>S. aureus</i>-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells.</p></div

Impairment of autophagic flux in <i>PRIP-</i>DKO MEFs.

<p>(A–C) Time-lapse analysis of <i>S. aureus</i>-infected MEFs. MEFs transiently transfected with the mRFP-GFP-LC3 plasmid were cultured with <i>S. aureus</i> (MW2) for 1.5 h, after which extracellular bacteria were killed by lysostaphin treatment. In wild-type (WT) cells (upper panels), a RFP(+)GFP(+)-LC3-labeled autophagosome (arrowhead) appeared at 3 h 30 min post-infection and changed to a RFP(+)GFP(−)-LC3-labeled autolysosome at 4 h 21 min post-infection (asterisk). In <i>PRIP</i>-DKO images (DKO, lower panels), an RFP(+)GFP(+)-LC3-labeled autophagosome (arrowhead) appeared at 4 h 30 min post-infection, and the RFP(+)GFP(+) signal (yellow) was not altered during the experiment (terminated at 6 h 24 min post-infection). The closed bars in graphs (B) represent the elapsed time for RFP(+)GFP(+) autophagosome formation in WT (upper) and <i>PRIP</i>-DKO (lower) MEFs. Asterisks in (B) indicate the experiment replicates corresponding to images in (A). The graph in (C) shows the proportion of the elapsed times (<120 min: dark shading, 120–180 min: hatched shading, and >180 min: no shading) in WT and <i>PRIP</i>-DKO cells.</p

Impairment of autophagosomal maturation in <i>PRIP</i>-DKO MEFs.

<p>(A, B) MEFs transiently expressing mRFP-GFP-LC3 were incubated with <i>S. aureus</i> (ATCC 29213) for 3 h (A) and 4.5 h (B). Images were obtained by confocal laser microscopy. A set of representative images from three independent experiments using wild-type (WT) and <i>PRIP</i>-DKO (DKO) MEFs are shown. Bacteria were stained with DAPI (blue). LC3-positive autophagosome-like vacuoles with RFP(+)GFP(−) signal indicate the formation of autolysosomes (arrowheads). Scale bars represent 20 µm in the left panel and 5 µm in the three right panels. (C) The graph shows the ratio of the number of <i>S. aureus</i> cells entrapped in RFP(+)GFP(−) vacuoles <i>vs.</i> the number of <i>S. aureus</i> cells entrapped in RFP(+) vacuoles. Values are expressed as means ±SEM (n = 60 cells for each genotype and each infection time from three independent experiments). WT, 5.4±1.3% (3 h) and 25.7±3.8% (4.5 h); DKO, 0.9±0.3% (3 h) and 3.7±1.3% (4.5 h). *<i>p</i><0.05, ***<i>p</i><0.001.</p