Expression of GFP and NeuN in rat DRG following intrathecal vector injection.

<p>A,B and C) GFP (green) and NeuN (red) were co-labeled in A) lumbar, B) thoracic and C) cervical DRG at 2 week-post vector injection. D) Bar graph represents size distribution of DRG neurons with and without GFP labeling. A total of 3781 neurons from the L4, L5 and L6 DRGs of 3 animals were included. Scale bar, 250 µm.</p

Expression of mTOR in lumbar DRGs following intrathecal vector injection.

<p>A) Schematic drawing of the AAV vector that encodes sh-RNA. The sequences of the two active and one control siRNA (si-Luc) are shown. B) Representative Western blots showing levels of mTOR and beta-tubulin in naïve rats or at two week following vector administration in the lumbar DRGs (L4 and L5 DRGs from both sides were pooled). C) Histogram represents the mean mTOR levels in respect to the naive group. The data are presented as mean ± SEM of 4–6 rats per group. D) Representative Western blots showing levels of mTOR and beta-tubulin at five week following vector administration in the lumbar DRGs (L4 and L5 DRGs from both sides were pooled). E) Histogram represents the mean mTOR levels in respect to the control group (si-Luc). The data are presented as mean ± SEM of 4 rats per group. F, G and H) Confocal images of mTOR (red), GFP (green) and NeuN (blue) in L5 DRG at two week following F) si-Luc, G) si-TOR and H) si-TOR' vector administration. Scale bar, 100 µm. PGK, phosphoglycerate kinase promoter; ITR, inverted terminal repeat; U6, U6 promoter.</p

Expression of GFP in different populations of neurons in the lumbar DRG.

<p>A,B C and D) GFP (green) was co-labeled with A) NF200, B) TRPV1, C) CGRP and D) IB4 (red) at two week following vector administration. Arrowheads indicate co-localization of GFP with the respective cell marker. E) Bar graph represents percentage of GFP-positive neurons co-labeled with the aforementioned markers. The data are presented as mean ± SEM of 3 rats. Scale bar, 100 µm.</p

Expression of mTOR in L5 DRG following intrathecal vector administration.

<p>A) Confocal images of mTOR (red) co-labeled with NF200, CGRP, TRPV1 or IB4 (green) in L5 DRG 2 weeks following the si-Luc or si-TOR vector injection. Double labeled neurons are indicated with asterisks. B) Bar graph represents percentage of mTOR-positive neurons co-labeled with the aforementioned markers. The data are presented as mean ± SEM of 3–4 rats except TRPV1 represents a single rat. C) GFP (green) and mTOR (red) in L5 DRG at 1 week following the si-Luc vector and 1, 2 and 5 weeks following the si-TOR vector injection. Scale bars, A, 50 µm and C, 100 µm.</p

Weight gain and nociceptive behaviors following the siRNA vectors.

<p>A) Daily weight gain of rats after receiving no vector (control), si-Luc or si-TOR vector. The mean weight gain in a two week period following vector injection is shown. B) Thermal and C) mechanical thresholds for naïve rats and rats treated with GFP, si-Luc or si-TOR vector for two weeks. D) Total number of flinches in phase I (1–9 min) and II (10–60 min) following intraplantar formalin (2.5%, 50 µL) injection. Rats received si-Luc or si-TOR vector two weeks before the formalin tests. E) Mechanical thresholds of the ipsilateral paw following unilateral L5 and L6 spinal nerve ligation. Rats received si-Luc or si-TOR vector two weeks before the SNL surgery. The data are presented as mean ± SEM of 6–8 rats per group.</p

DRG ATF3, Iba1 immunohistochemistry and sciatic nerve myelin staining following intrathecal vector injection.

<p>A) Expression of ATF3 in the L5 DRGs in rats treated with spinal nerve ligation (SNL, 2 weeks), si-Luc or si-TOR AAV5 vectors (4 weeks). B) Myelin staining of the sciatic nerve from rats treated with si-Luc, si-TOR or si-TOR' vectors (4 weeks). C) Expression of IbaI in the L5 DRGs in a naïve rat or rats received si-Luc, si-TOR or GFP-only vectors. A cervical DRG from a rat treated with si-TOR vector was also shown. Scale bar, 100 µm.</p

Combinations of pSP-D-CD40L, CpG, and poly(I:C) showed strong antitumor effects on established B16F10 melanoma.

<p>Given the promising data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007334#pone-0007334-g002" target="_blank">Fig. 2</a>, further studies were done to determine the relative contributions of pSP-D-CD40L, CpG, and poly(I:C) and the effects of using them in a triple combination. Twelve groups of mice (5/group) were studied in parallel. For display purposes, the data are grouped into three rows of graphs focusing on CpG (top row), poly(I:C) (middle row), and CpG + poly(I:C) (bottom row). Panels A, B, and C – While each individual agent slowed tumor growth, the most significant antitumor effect was produced by the combination of pSP-D-CD40L + CpG + poly(I:C). Panel A shows that CpG alone significantly slowed tumor growth compared to either PBS or pcDNA3.1 alone from day 12 (p<0.01 by Student's t test, mean±SEM, n = 5). In this fully controlled experiment, however, it was clear that the addition of pSP-D-CD40L to CpG produced no further antitumor effects (p>0.05). Similarly, Panel B shows that poly(I:C) alone significantly slowed tumor growth when compared to PBS or pcDNA3.1 alone from day 12 (p<0.01). Again, however, the combination of pSP-D-CD40L + poly(I:C) produced no further antitumor effects (p>0.05). Interestingly, as shown in Panel C, the double combination of CpG + poly(I:C) significantly reduced tumor growth beyond that produced by CpG alone (p<0.05 on day 24 on the combination as compared to CpG alone). The addition of pSP-D-CD40L to the two TLR agonists, CpG and poly(I:C), produced an even stronger antitumor effect (Panel C, p<0.05 on day 24 comparing the triple combination to CpG + poly(I:C)). Panels D, E, and F – For survival, the addition of pSP-D-CD40L did not increase the antitumor effects seen with CpG alone. All three agents (pSP-D-CD40L, CpG, and poly(I:C)) improved survival as single therapies. From pairwise comparisons, the survival benefit was greatest with CpG and less prominent with pSP-D-CD40L and poly(I:C). The combination of CpG + poly(I:C) improved survival further compared to poly(I:C) alone (p<0.05 by log-rank test). Although the effects on tumor growth indicated that the double combination of TLR agonists CpG + poly(I:C) was better than each alone, this was not reflected in the survival data. Similarly, the superiority of the triple combination of pSP-D-CD40L + CpG + poly(I:C) seen in the tumor growth studies was not statistically significant from the survival data.</p

Tumor-dependent differences in the immunohistology of induced tumor regression.

<p>Panel A – Histology of control and treated tumors. Tumors were injected every other day X 5 with PBS as a control or with the triple combination of pSP-D-CD40L + CpG + poly(I:C). As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007334#pone-0007334-g003" target="_blank">Figure 3</a>, the triple combination slowed the growth of tumors, and occasionally led to tumor eradication. Two days after the last injection, tumor tissue was processed for histology by staining with hematoxylin and eosin. Tumors treated with PBS showed areas of spontaneous necrosis suggesting that the rapidly growing tumor cells often outgrow their blood supply. After treatment with the triple combination, large areas of necrotic tissue appeared containing fragmented cells and nuclear remnants consistent with a cell death process that exceeded the availability of phagocytic macrophages to clear the debris (see Panel D). Panel B – CD11c antibody staining for dendritic cells. B16F10 tumors injected with PBS as a control contained identifiable CD11c+ dendritic cells. After treatment with the triple combination, even fewer dendritic cells were found in the tumors. Panel C – CD8 antibody staining. For tumors injected with PBS as a control, relatively few CD8+ T cells were seen. However, following injections with the triple combination, there was a marked increase in intratumoral CD8+ T cells in all tumor sections examined. Panel D – F4/80 antibody staining for macrophages. Tumors injected with PBS as a control contained relatively few F4/80+ macrophages and there was no appreciable increase in F4/80+ macrophages following treatment with the triple combination.</p

PEI nanoparticle delivery of pSP-D-CD40L slowed tumor growth and prolonged survival.

<p>The data shown are representative of three independent experiments. Panel A – Antitumor effects of PEI plasmid DNA nanoparticles prepared with pSP-D-CD40L alone or in combination with CpG or CpG + poly(I:C). The role of DNA transfection efficiency was tested by preparing nanoparticles formed from PEI and pSP-D-CD40L plasmid DNA. Intratumoral injections of PEI pSP-D-CD40L nanoparticles led to significantly slower tumor growth (p<0.05 on day 10) when compared to the injection of naked pSP-D-CD40L plasmid alone. Panel B – Survival benefit of PEI pSP-D-CD40L nanoparticle injections in combination with CpG + poly(I:C). As expected from the tumor growth data, pSP-D-CD40L formulated with PEI was able to enhance mouse survival when combined with CpG and poly(I:C) TLR agonists. This combination therapy resulted in long-term-tumor free survival of 2/5 mice (p<0.01 compared to pcDNA3.1)).</p

Increase in Hoffmann reflex and loss of rate-dependent depression (RDD) in spinally transected rats at 3 months after transection.

<p><b>(A, B)</b>—Measurement of H-reflex and statistical analysis of the H/M ratio showed a significant increase in responses at 3 months after transection if compared to wild-type non-injured animals (unpaired two-tailed t-test; ***P< 0.001). <b>(C)</b>—Testing of RDD showed a significant loss of RDD in spinally-transected animals at stimulation frequencies of 1, 5 and 10 Hz (one-way ANOVA; Bonferroni post hoc; ***P< 0.001).</p