Representative pictures of rat testes at 12 weeks post-irradiation.

<p>Radiation caused the rat testes to shrink in size (∼60%, PBS RAD) as compared to non-irradiated rat testes (Control PBS). However, testes from animals treated with MnTE-2-PyP (MnTE-2-PyP RAD) were indistinguishable from non-irradiated testes.</p

MnTE-2-PyP protected from irradiation-induced skin damage.

<p>A. Representative pictures of lower abdomens at 6 weeks post-irradiation. Irradiation caused marked epilation, which MnTE-2-PyP protected. B. Irradiation caused erythma and moist desquamation in the exposed skin exposed. The skin of irradiated rats injected with MnTE-2-PyP, had significantly less severe erythma and no moist desquamation. These skin changes persisted through the course of the experiment, n = 8 rats per group, asterisk (*) denotes p<0.05.</p

Diagram of Experimental Design.

<p>Rats received 5 consecutive radiation doses of 7.5 Gy to the lower abdomen. Rats were injected i.p. with MnTE-2-PyP or PBS as a control throughout the study as indicated. Animals were harvested 12 weeks post-irradiation. There were four animals per group and the experiment was repeated once.</p

A model demonstrating how MnTE-2-PyP could work as a radioprotectant.

<p>We have shown that the potent antioxidant, MnTE-2-PyP protects normal tissues from irradiation-induced damage. We hypothesize that MnTE-2-PyP can inhibit injury of healthy bystander tissues by inhibiting inflammation driven by oxidative stress. We also hypothesize that MnTE-2-PyP has no effect on the killing of tumor cells due to the inability of MnTE-2-PyP to scavenge the damaging hydroxyl radical release caused by irradiation. Thus, MnTE-2-PyP can protect normal tissue from irradiation damage while not compromising the ability of irradiation to effectively kill tumor cells.</p

MnTE-2-PyP reduces DNA oxidative damage induced by irradiation in prostate epithelial cells.

<p>Irradiation is a known inducer of 8-OHdG, a marker of DNA oxidative damage. MnTE-2-PyP significantly reduced 8-OhdG in the prostate of irradiated animals 6 weeks post-irradiation. n = 4 rats/group, asterisk (*) denotes a significant difference from the RAD group.</p

Irradiated rats receiving MnTE-2-PyP lost significantly less weight than irradiated rats injected with PBS.

<p>Weights of irradiated animals throughout the course of the experiment as compared to their respective non-irradiated groups. n = 8 rats per group, asterisk (*) denotes p<0.05.</p

Pharmacokinetics of MnTE-2-PyP in the rat urogenital system.

<p>Rats received injections of MnTE-2-PyP as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044178#pone-0044178-g001" target="_blank">Figure 1</a> and harvested 1 week, 2 weeks, 12 weeks and 12 weeks+1 day after the start of injections. Rats harvested at 1 and 2 weeks had not received an injection for 3 days (3 day trough). Rats harvested at 12 weeks, had not received an injection for 7 days (representing the lowest levels during the week) and 12 weeks+1 day represent rats 1 day after an injection (representing the highest levels during the week). A. Liver. B. Bowel. C. Prostate. D. Penile tissues. E. Bladder. MnTE-2-PyP was detected in the liver, bowel, prostate, penile tissue and bladder. Data represents the mean ± standard error of the mean, n = 3 per time point.</p

Inhibition of Nuclear Factor-Kappa B Activation Decreases Survival of <i>Mycobacterium tuberculosis</i> in Human Macrophages

<div><p></p><p>Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to <i>Mycobacterium tuberculosis</i> (<i>MTB</i>). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to <i>MTB</i>. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular <i>MTB</i>. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular <i>MTB</i> in human macrophages via induction of apoptosis and autophagy.</p></div

Inhibition of NFκB activation induces autophagy in THP-1 cells.

<p>(<b>A</b>) Control THP-1 cells (0.1% DMSO) and THP-1 cells subjected to serum starvation, 5 µM BAY, <i>MTB</i> infection, or both <i>MTB</i>+BAY for 24 hrs, followed by immunoblotting of nuclear-free whole cell lysates for LC3 and β-actin. A representative immunoblot of three independent experiments is shown. (<b>B</b>) Human THP-1 cells were transduced with lentivirus-GFP-LC3 and differentiated into macrophages, followed by infection with <i>MTB</i> for 24 hrs in the absence or presence of 5 µM BAY. The cells were fixed and stained with DAPI to visualize the nuclei (blue) and the number of GFP-positive punctae were quantified. <i>Upper panel</i>, representative immunofluorescence images of three independent experiments; <i>lower panel</i>, average number of GFP-LC3 punctae per cell. The data shown represent the mean ± SEM of duplicate wells/condition from three independent experiments. (<b>C</b>) <i>MTB</i>-infected THP-1 cells treated with BAY were incubated with or without 3-MA, an inhibitor of the early phase of the autophagic pathway. After 48 hrs, the cells were lysed and nuclear-free whole cell lysates (20 µg per lane) were separated by SDS-PAGE and immunoblotted for LC3-I, LC3-II and β-actin. The bar graph represents the relative densities of the LC3-II bands normalized for their corresponding β-actin bands for two independent experiments. (<b>D</b>) THP-1 cells were infected with <i>MTB</i> H37Rv alone, <i>MTB</i>+5 µM BAY, or <i>MTB</i>+BAY+6 mM 3-MA for 4 days and cell-associated <i>MTB</i> was quantified. Data shown are mean ± SEM from two independent experiments performed in duplicates. *p<0.05, **p<0.01, ***p<0.001.</p

Diagram of the mechanisms by which NFκB activation promotes the intracellular survival of <i>MTB</i>.

<p>Based on our experimental findings, NFκB activation enhanced the intracellular survival of <i>MTB</i> through inhibition of apoptosis and autophagy in infected macrophages. Since NFκB can also induce the production of its inhibiting molecule IκBα (blue line) and NFκB inhibition of autophagy could potentially prevent degradation of IKK (red line), the ultimate effect of NFκB on survival of intracellular <i>MTB</i> in macrophages is likely a complex process. IKK = IκBα kinase.</p