Amino acid sequence of cSN50.1 peptide and its congeners SN50 and cSN50.

<p>Fragment-linked peptides comprising the Signal Sequence Hydrophobic Region of Fibroblast Growth Factor 4 (bolded) and the NLS region of NFκB1/p50 (italicized) were analyzed for their solubility in water. In cSN50 and cSN50.1, an intra-molecular disulfide bond is formed between the two cysteines, which cyclizes the NLS motif.</p><p>Amino acid sequence of cSN50.1 peptide and its congeners SN50 and cSN50.</p

LPS-induced cellular trafficking to lungs is reduced in NTM-treated mice.

<p>Cell counts in BAL fluid collected from unchallenged mice (naïve) and 6 h after direct airway exposure to LPS, with i.p. NTM peptide (cSN50.1) or diluent control (saline) treatment. The LPS-induced increase in total cells is comprised primarily of neutrophils. Neutrophil trafficking to BAL is significantly reduced by NTM treatment while monocytes/macrophages and lymphocytes are not affected. Data are presented as mean ± standard error, <i>n</i> = 4 naïve and 5–7 NTM- or saline-treated animals/group from two independent experiments. *<i>p</i><0.05, **<i>p</i><0.005 by Mann-Whitney <i>U</i> test comparing LPS-challenged groups.</p

Genes encoding mediators of inflammation are regulated by transcription factors that require nuclear transport shuttles targeted by NTM.

<p>Analysis was performed <i>in silico</i> using The UCSC Genome Browser (The UCSC Genome Bioinformatics). mNLS - classical monopartite Nuclear Localization Sequence; bNLS - nonclassical bipartite Nuclear Localization Sequence; uNLS - unconventional, structural or dimer-specific Nuclear Localization Sequence; bHLH - basic helix-loop-helix motif; “+” - the transcription factor specific binding site is present in the gene promoter.</p><p>Genes encoding mediators of inflammation are regulated by transcription factors that require nuclear transport shuttles targeted by NTM.</p

NTM treatment enhances survival from lethal endotoxic shock.

<p>Survival curves for mice challenged i.p. with high-dose LPS (A and B) or low-dose LPS+D-Gal (C). In (A) and (C) mice were administered the first NTM (cSN50.1 peptide) treatment 30 min before LPS challenge (prophylactic protocol), while in (B), the first treatment was administered 15 min after LPS challenge (therapeutic protocol). Saline injections were administered to control mice challenged with LPS following the same treatment schedule in each protocol, as described in “Materials and Methods”. ***<i>p</i><0.0005 by log rank test.</p

NTM treatment reduces plasma levels of multiple cytokines, chemokines and growth factors induced by LPS.

<p>(A) Wild type C57BL/6 mice were challenged i.p. with a lethal dose of LPS (800 µg) and treated with i.p. injections of NTM (cSN50.1 peptide) or diluent (saline) following a prophylactic protocol as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110183#pone-0110183-g004" target="_blank">Figure 4A</a>. Blood was collected at baseline and 2 or 6 h after LPS challenge and a multiplex assay was used to measure 32 analytes in plasma. Twenty-four analytes were significantly altered by NTM treatment, as determined by repeated measures two-way analysis of variance with Sidak’s post-test. Twenty-three were reduced, while anti-inflammatory IL-10 was increased by NTM treatment. Results are shown as the % inhibition or increase by NTM compared to saline control set to 100% at the time point demonstrating maximal expression for that analyte. <i>n</i> = 10 animals/group. (B) Comparison of prophylactic and therapeutic NTM treatment protocols on selected plasma cytokine and chemokine levels in the high-dose LPS model of endotoxic shock. Data are presented as mean ± standard error, <i>n</i> = 5 −10 animals/group.</p

LPS-induced expression of chemokines, cytokines, and growth factors in the lung is suppressed by NTM.

<p>Fourteen cytokines, chemokines and growth factors elevated in BAL after direct airway exposure to LPS are significantly suppressed by NTM (cSN50.1 peptide) treatment. Data are presented as mean ± standard error, <i>n</i> = 4 naïve and 8–9 NTM- or saline-treated animals/group from 3 independent experiments. *<i>p</i><0.05, **<i>p</i><0.005, and ***<i>p</i><0.0005 by Mann-Whitney <i>U</i> test comparing LPS-challenged groups.</p

Survival rates and bacterial loads in organs of intranasally immunized BALB/c mice after intranasal challenge with <i>B</i>. <i>thailandensis</i> (5 x 10⁶ CFU).

<p><b>(A)</b> Mice were immunized with PBS, SL3261 (vector), or SL3261/p1C3 (vaccine) via the intranasal route, then challenged. Mice were monitored for survival for a period up 150 h post-challenge. Results are represented in Kaplan-Meier survival curves and were analyzed by log-rank test. Log-rank: PBS vs. vaccine (<i>P =</i> 0.0031); vector vs. vaccine (<i>P =</i> 0.0128). Median survival: PBS, 64 h; SL3261, 84 h; and SL3261/p1C3, undefined). Results are representative of three independent experiments, five mice each. <b>(B, C)</b> Bacterial load in the organs of intranasally immunized mice 72 h post-challenge. All samples were plated for viable CFU on TSA and Ashdown’s agar. Each point represents a single mouse. ND indicates not detected. Data was analyzed by the Mann-Whitney <i>U</i> test. Nasal wash (<i>P</i> = 0.0012 vaccine vs. vector and <i>P</i> = 0.0025 vaccine vs. PBS); lung (<i>P</i> = 0.0043 vector vs. PBS); liver (<i>P</i> = 0.0043 vector vs. PBS).</p

Opsonophagocytosis killing of <i>B</i>. <i>thailandensis</i>.

<p><i>In vitro</i> opsonophagocytic killing of <i>B</i>. <i>thailandensis</i> E264<i>lux</i> using dilutions of pooled antisera collected from intranasally PBS-, SL3261 (vector)-, and SL3261/p1C3 (vaccine)-immunized BALB/c mice. Plates were read at 120 min following the co-incubation of the opsonophagocytosis assay components. Bars represent mean percentage of killing, and error bars represent the standard deviation. Data were analyzed by the Mann-Whitney <i>U</i> test (<i>P<0</i>.<i>0005</i> vaccine vs. no sera; <i>P<0</i>.<i>005</i> vaccine vs. vector).</p

Serum antibody response of BALB/c mice following intranasal immunization with <i>S</i>. <i>enterica</i> serovar Typhimurium SL3261 expressing <i>B</i>. <i>mallei</i> O antigen.

<p>Sera were collected 4 weeks post-vaccination, and the data were analyzed by the Mann-Whitney <i>U</i> test. Serum IgG and IgM response to: <b>(A)</b><i>B</i>. <i>mallei</i> LPS (<i>P</i> < 0.0001 vector vs. vaccine for IgG and IgM); <b>(B)</b><i>B</i>. <i>pseudomallei</i> LPS (<i>P</i> < 0.0003 vector vs. vaccine for IgG, and <i>P</i> < 0.0079 for IgM); <b>(C)</b><i>B</i>. <i>thailandensis</i> E264 lysate (<i>P</i> < 0.005 PBS vs. vaccine, and <i>P</i> < 0.0005 vector vs. vaccine). <b>(D)</b> Specific IgG response in the sera of intranasally-immunized BALB/c mice to <i>B</i>. <i>mallei</i> LPS or anti-<i>B</i>. <i>thailandensis</i>.</p

Serum cytokine response of BALB/c mice following intranasal immunization with <i>S</i>. <i>enterica</i> serovar Typhimurium SL3261 expressing <i>B</i>. <i>mallei</i> O antigen.

<p>Sera were collected 4 weeks post-vaccination, and the data were analyzed by the Mann-Whitney <i>U</i> test. <i>P</i> values were calculated as: Vaccine vs. PBS and Vector vs. PBS.</p