Ang II inhibits <i>P</i>. <i>falciparum</i> growth <i>in vitro</i> and <i>P</i>. <i>berghei</i> growth in mice.

<p>A) Parasite levels in cultures of <i>P</i>. <i>falciparum</i> incubated with different concentrations of Ang II <i>in vitro</i>. Ratio of day 0 (initial parasitemia) to day 1 (right axis, circles) and percentage rupture of infected erythrocytes (left axis, squares). B) Parasitemia was determined in groups of mice that were infected with <i>P</i>. <i>berghei</i> (day 0) and had been subjected (n = 8; circles) or not (n = 8; squares) to implantation of subcutaneous mini-pumps releasing saline buffer. C) Parasitemia of groups of mice infected with <i>P</i>. <i>berghei</i>: control (n = 14; circles), implanted with intradermal micro-pumps releasing Ang II at 100 ng/kg/min (n = 15; squares) or 500 ng/kg/min (n = 15; triangles). Number of surviving mice in each group are indicated in the table. Mice numbers decrease due to experimental cerebral malaria induced death (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138191#pone.0138191.g002" target="_blank">Fig 2B</a>). (B,C) Statistical analysis by t-Student (B) and Kruskal Wallis (C). Average plus standard error is shown. * <i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

Ang II decreases brain hemorrhages, delays experimental cerebral malaria and increases survival of mice.

<p>Groups of mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138191#pone.0138191.g001" target="_blank">Fig 1C</a>, control (black circles); Ang II at 100 and 500 ng/kg/min (black squares and triangles, respectively) were observed twice a day for determination of cerebral malaria based on neurological symptoms (as described in methods) (A) and survival (time of euthanasia) (B). Kaplan-Meier analyses were conducted to determine differences among the groups. Cerebral malaria and survival distribution of the groups were significantly different (<i>p</i><0.010 and <i>p</i><0.013, respectively). Pairwise log rank comparison after applying Bonferroni correction (statistical significance accepted at the <i>p</i><0.0167 level) showed that for cerebral malaria there was a statistically significant difference between control <i>vs</i> Ang II (100) (<i>p</i> = 0.0021) but not <i>vs</i> Ang II (500) (<i>p</i> = 0.0258). For survival, both Ang II groups showed significant differences vs control group (<i>p</i> = 0.0087 and <i>p</i> = 0.0100 for Ang II (100) and Ang II (500) <i>vs</i> control, respectively). No significant differences were found between angiotensin groups, Ang II (100) and Ang II (500) (<i>p</i> = 0.511 and <i>p</i> = 0.524 for cerebral malaria and survival respectively). Histological sections of the brains of mice control (C), Ang II at 100 ng/kg/min (D) and Ang II at 500 ng/kg/min (E) were analyzed for the presence and size of hemorrhages. Bar is 50μm. Quantification of the number (F) and size (G) of hemorrhages per mm². Mice that were sacrificed with a cerebral malaria score of 3 (black circles), 4 (blue circles) or 5 (red circles) and mice that did not develop cerebral malaria (gray circles) are indicated. Average values for each mouse are shown. **<i>p</i><0.01 by ANOVA. No significant differences were observed between groups in (G).</p

Paricalcitol reduced peritoneal membrane fibrosis, inflammation and ultrafiltration failure in mice exposed to PDF.

<p>A) Paraffin sections of the peritoneal membrane from the 3 groups were stained with Masson's trichrome. B) Thickening of the peritoneal membrane was determined by morphometric analysis. C) Peritoneal permeability was determined by net ultrafiltration. D) The presence of inflammatory and mesothelial cells was determined by the expression of CD45 (green) and cytokeratin (red), respectively, in frozen sections of peritoneal membrane representative of each group. A green arrow indicates hematopoietic cells. A red arrow indicates mesothelial cells. E) The angiogenesis was determined by the expression of CD31 (green). Cytokeratin-positive cells are stained in red. The color balance was equally adjusted in immunofluorescence using Photoshop V10 for Mac (Abobe Systems Incorporated, US). n≥5 in each group. Statistical significance was determined using the Mann-Whitney test. *<i>P</i><.05; ***<i>P</i><.001.</p

Paricalcitol induced the recruitment of regulatory CD8+ T cells into the peritoneal cavity.

<p>Paricalcitol (dashed box) tended to increase the frequency of CD4⁺ T cells expressing Foxp-3 (A), but not CTLA-4 (B) or membrane TGF-β (C). Paricalcitol increased the frequency of CD8⁺ T cells expressing Foxp-3 (A), CTLA-4 (B) and membrane TGF-β (C) compared with the PDF-group (white box). n≥9 in each group. Paricalcitol treated group had higher number of CD4⁺ CD8⁺ T cells expressing Foxp-3 than PDF group (D). Statistical significance was determined using the Mann-Whitney test. *<i>P</i><.05; **<i>P</i><.01; ***<i>P</i><.001.</p

Chemokine concentrations in the peritoneal cavity were not affected by paricalcitol treatment.

<p>Concentrations of RANTES (A), MIP-1α (B) and MIP-1β (C) were determined in the peritoneal washing fluid. We observed no difference between the paricalcitol and the PDF group. n≥5 in each group.</p

TWEAK induces apoptosis in serum depleted androgen-independent PC-3 cells but not in androgen-sensitive LNCaP cells.

<p>Cell death was assessed by flow cytometry of DNA content. Hypodiploid cells were considered apoptotic. A) PC-3 cells, cultured with or without 10% FBS, were stimulated with TWEAK (100 ng/mL) alone or in presence of TNFα (30 ng/mL)/IFNγ (30 U/mL) for 24 hours. Mean (±SD) of four independent experiments. *p<0.002 vs control; #p<0.001 vs TWEAK alone; †p<0.002 vs 0% FBS with stimuli. B) LNCaP cells, cultured without FBS, were stimulated with TWEAK alone or in combination with TNFα/IFNγ for 24 hours. Mean (±SD) of three independent experiments. C) TWEAK, alone or in combination with TNFα/IFNγ, induces apoptosis in PC-3 cells in a dose-dependent manner. Mean (±SD) of three independent experiments. *p<0.002 vs control; #p<0.0001 vs control. D) Time-course of TWEAK-induced apoptosis in PC-3 cells, alone or in combination with TNFα/IFNγ. Mean (±SD) of three independent experiments. *p<0.002 vs control; #p<0.0001 vs control.</p

Paricalcitol changed the peritoneal T cell population in mice instilled with PDF.

<p>A) The paricalcitol group tends to increase the number of nucleated cells at the peritoneal cavity compared with the PDF group. B) The number of B cells was similar in the PDF and paricalcitol groups. C) There is no significant difference between the number of macrophages in the PDF and paricalcitol groups. D) Paricalcitol increased the numbers of CD4⁺ T cells compared with the PDF group. E) The treatment with paricalcitol increased CD8⁺ T cells in the peritoneal cavity. Statistical significance was determined using the Mann-Whitney test. ***<i>P</i><.001; n≥5 in each group.</p

Paricalcitol induced the regulation of IL-17 production and affected peritoneal fibrosis outcomes.

<p>A) Peritoneal concentrations of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-γ and TNF-α were not affected by paricalcitol treatment. B) Paricalcitol induced the suppression of IL-17 production in the peritoneal membrane. C) The IL-17 concentrations strongly correlate with peritoneal fibrosis. n≥5 in each group. Statistical significance was determined using the Mann-Whitney test. **<i>P</i><.01. A correlation analysis was performed using Spearman's nonparametric test.</p

TWEAK induces cytochrome C release from the mitochondria in PC-3 cells.

<p>PC-3 cells treated with for 24 hours showed mitochondrial cytochrome C release. Confocal microscopy. Cytochrome C in red and DAPI in blue. (Magnification x400, detail x1000). Pictures representatives of three experiments.</p

TWEAK-induced PC-3 cell death has features of apoptosis. A)

<p>Flow cytometry of DNA content. Note hypodiploid, apoptotic cells (|――|) among those treated with TWEAK, or with TWEAK/TNFα/IFNγ for 24 hrs. Mean (±SD) of four experiments.*p<0.05 vs control. <b>B)</b> Characteristic shrunk, pyknotic, fragmented nuclei are present among DAPI-stained, TWEAK or TWEAK/TNFα/IFNγ-treated cells (arrows), but are infrequent among control or TNFα/IFNγ-treated cells. Magnification x400, detail x1000. <b>C)</b> PC-3 cells were cultured with or without serum and treated with TWEAK or TWEAK/TNFα/IFNγ for 24 hours. Cells were staining with Annexin V/PI and analyzed by flow cytometry. TWEAK increases both early (AnnexinV⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptosis under serum deprivation conditions. Graphic shows percentage of early and late apoptosis [(AnnexinV⁺/PI⁻)+(AnnexinV⁺/PI⁺)]. Mean (±SD) of three independent experiments. *p<0.008 vs control; #p<0.001 vs 0% FBS with stimuli.</p