Survival rates of infected mice.

<p>After intravenous inoculation with 3 × 10⁷ CFU of <i>S. aureus</i>, the survival rates of WT mice and TLR2-deficient mice were determined over a 14-d experimental period. Survival curves were generated from 2 independent experiments, with a total of 20 mice per group.</p

Imaging Mass Spectrometry Revealed the Accumulation Characteristics of the 2-Nitroimidazole-Based Agent “Pimonidazole” in Hypoxia

<div><p>Hypoxia, or low oxygen concentration, is a key factor promoting tumor progression and angiogenesis and resistance of cancer to radiotherapy and chemotherapy. 2-Nitroimidazole-based agents have been widely used in pathological and nuclear medicine examinations to detect hypoxic regions in tumors; in particular, pimonidazole is used for histochemical staining of hypoxic regions. It is considered to accumulate in hypoxic cells via covalent binding with macromolecules or by forming reductive metabolites after reduction of its nitro group. However, the detailed mechanism of its accumulation remains unknown. In this study, we investigated the accumulation mechanism of pimonidazole in hypoxic tumor tissues in a mouse model by mass spectrometric analyses including imaging mass spectrometry (IMS). Pimonidazole and its reductive metabolites were observed in the tumor tissues. However, their locations in the tumor sections were not similar to the positively stained areas in pimonidazole-immunohistochemistry, an area considered hypoxic. The glutathione conjugate of reduced pimonidazole, a low-molecular-weight metabolite of pimonidazole, was found in tumor tissues by LC-MS analysis, and our IMS study determined that the intratumor localization of the glutathione conjugate was consistent with the area positively immunostained for pimonidazole. We also found complementary localization of the glutathione conjugate and reduced glutathione (GSH), implying that formation of the glutathione conjugate occurred in the tumor tissue. These results suggest that in hypoxic tumor cells, pimonidazole is reduced at its nitro group, followed by conjugation with GSH.</p></div

Kinetics of the bacterial load in murine organs.

<p>After infection with <i>S. aureus</i>, the counts of viable bacteria present in the livers (A) and kidneys (C) of WT and TLR2-deficient mice were determined from 1 to 5 d. Each point represents the mean ± SD of the <i>S. aureus</i> CFU from 2 independent experiments, with a total of 20–30 mice per group. ^#<i>p</i> < 0.01 vs. infected WT mice. (B) Representative Gram-stained photomicrographs (original magnification, × 100) of livers from uninfected mice and infected mice at 3 d post-infection are shown.</p

CD36 expression in mouse macrophages.

<p>Following treatment with heat-killed <i>S. aureus</i> (MOI of 5), the macrophages from WT and TLR2-deficient mice were examined for CD36 mRNA and protein expression by real-time PCR and laser-scanning confocal microscopy. Untreated mouse macrophages served as a control. (A) CD36 was present in the plasma of macrophages from both WT and TLR2-deficient mice under the no-treatment condition. Confocal microscopy showed rapid CD36 localization on the plasma membrane of the macrophages from WT mice at 5 min post-treatment; this expression rapidly disappeared thereafter. No change was observed in the cells from TLR2-deficient mice (original magnification, × 400). (B) The time course of CD36 mRNA expression in the 2 macrophage groups is shown. Results were normalized to GAPDH gene expression and are shown as fold changes relative to gene expression in the control cells. Data are expressed as the mean ± SD for 3 separate experiments.</p

Representative mass spectrometric images of pimonidazole and its low-molecular mass metabolites and pimonidazole immunohistochemical staining in mouse tumors 0.5, 2 and 4 h after administration of pimonidazole.

<p>The scale bar represents 1 mm. (A)–(F): Mass spectrometric images of mouse tumor 0.5 h after administration. (G)–(L): Mass spectrometric images of mouse tumor 2 h after administration. (M)–(R): Mass spectrometric images of mouse tumor 4 h after administration. (A), (G), (M): Mass spectrometric images of m/z 255.145, representing pimonidazole (<u><i>1</i></u>). (B), (H), (N): Mass spectrometric images of m/z 225.171, representing amino-pimonidazole (<u><i>4</i></u>). (C), (I), (O): Mass spectrometric images of m/z 239.150, representing nitroso-pimonidazole (<u><i>2</i></u>). (D), (J), (P): Mass spectrometric images of m/z 241.166, representing hydroxylamino-pimonidazole (<u><i>3</i></u>). (E), (K), (Q): Mass spectrometric images of m/z 530.239, representing the glutathione conjugate of amino-pimonidazole (<u><i>6</i></u>). (H), (L), (R): pimonidazole immunohistochemical staining.</p

Inflammatory infiltration and CD36 expression in mouse livers.

<p>To identify the infiltrating cell types and CD36 expression in the livers of infected WT mice and TLR2-deficient mice, murine liver tissues were immunostained for neutrophils, macrophages, and CD36, and analyzed for the CD36 gene expression level by real-time PCR. Representative photomicrographs (original magnification, × 200) of liver sections stained with an anti-MOP antibody for neutrophils at 6 h post-infection (A); an F4/80 antibody for macrophages at 2 d post-infection (B); and an anti-CD36 antibody for CD36 protein expression at 2 d post-infection (C); and the percentage of positively stained cells and the percentage of positively stained field areas are shown. Uninfected WT mice and TLR2-deficient mice served as controls. (D) Results from real-time PCR were normalized to GAPDH gene expression and are shown as fold changes relative to gene expression in the control mice. Data are presented as the mean ± SD for 5–7 mice per infected group and for 3 mice per uninfected group. ^*<i>p</i> < 0.05, ^#<i>p</i> < 0.01, ^$<i>p</i> < 0.001 vs. uninfected WT mice; ^∮<i>p</i> < 0.05, ^∞<i>p</i> < 0.01, ^§<i>p</i> < 0.001 vs. uninfected TLR2-deficient mice.</p

Phagocytosis of <i>S. aureus</i> by mouse macrophages.

<p>The peritoneal macrophages from WT and TLR2-deficient mice were left untreated or were treated for 60 min with Alexa Fluor 488-labeled <i>S. aureus</i> (MOI of 1 or 5) at 37°C and then harvested for flow cytometry analysis. (A) Histograms represent fluorescence intensity counts of 10,000 cells for each sample, and representative histograms are shown. (B) The percentage of positive cells that had engulfed bacteria is indicated. Data are expressed as the mean ± SD for 3 separate experiments.</p

Cytokine production in the livers (A), blood (B), and kidneys (C) of mice.

<p>After inoculation with viable <i>S. aureus</i> or PBS, the concentrations of TNF-α, IL-6, and IL-10 in organ extracts and plasma from WT mice and TLR2-deficient mice at the indicated time intervals were assayed by ELISAs. Data are the mean ± SD from 3 independent experiments, with a total of 20–30 mice per infected group and a total of 6 mice per uninfected group. ^*<i>p</i> < 0.05, ^#<i>p</i> < 0.01, ^$<i>p</i> < 0.001 vs. infected WT mice.</p

Combined Plasma and Tissue Proteomic Study of Atherogenic Model Mouse: Approach To Elucidate Molecular Determinants in Atherosclerosis Development

Atherogenic cardiovascular diseases are the major cause of mortality. Prevention and prediction of incidents is important; however, biomarkers that directly reflect the disease progression remain poorly investigated. To elucidate molecular determinants of atherogenesis, proteomic approaches are advantageous by using model animals for comparing changes occurring systematically (bloodstream) and locally (lesion) in accordance with the disease progression stages. We conducted differential mass spectrometric analysis between apolipoprotein E deficient (apoED) and wild-type (wt) mice using the plasma and arterial tissue of both types of mice obtained at four pathognomonic time points of the disease. A total of 100 proteins in the plasma and 390 in the arterial tissues were continuously detected throughout the four time points; 29 were identified in common. Of those, 13 proteins in the plasma and 36 in the arterial tissues showed significant difference in abundance between the apoED and wt mice at certain time points. Importantly, we found that quantitative variation patterns regarding the pathognomonic time points did not always correspond between the plasma and arterial tissues, resulting in gaining insight into atherosclerotic plaque progression. These characteristic proteins were found to be components of inflammation, thrombus formation, and vascular remodeling, suggesting drastic and integrative alteration in accordance with atherosclerosis development

Regression analyses of radiotracer accumulation levels and intraplaque macrophage infiltration/number of apoptotic cells.

<p>Positive correlations were observed between the ¹⁴C-FDG accumulation level and macrophage infiltration (Mac-2-positive areas) (<b>A</b>), and between the ^99mTc-annexin A5 accumulation level and apoptotic cell number (TUNEL-positive cells) (<b>B</b>).</p