Intracellular MPO activity correlates well with tissue neutrophil content.

<p>(<b>A</b>) Flow cytometry demonstrates different neutrophil counts in brain, heart, liver, spleen, and bone marrow, as quantified in (<b>B</b>) (n = 2 per group). (<b>C</b>) Intracellular MPO activity was measured with the antibody-capture assay using ADHP, and shows a similar trend to neutrophil content per organ (n = 2 per group). (<b>D</b>) A close correlation was found between neutrophil content and intracellular MPO activity in these organs. MPO = myeloperoxidase. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine.</p

<i>In vivo</i> imaging and <i>in vitro</i> MPO activity assays demonstrate markedly different findings.

<p>(<b>A</b>) MPO-Gd molecular MR imaging reveals MPO inhibition in vivo in mice with experimental autoimmune encephalomyelitis that were treated with ABAH. MPO activity maps are shown in 3D from two angles (left), as well as overlays of MPO activity maps over T1 images (right). (<b>B</b>) Quantification of imaging reveals significant difference in MPO activity <i>in vivo</i> (<i>P</i> = 0.03, n = 8 per group). (<b>C</b>) <i>In vitro</i> assays on whole tissue homogenates using ADHP or TMB do not confirm the <i>in vivo</i> imaging finding (<i>P</i> = 0.68 and 0.88, respectively, n = 4 per group). *: <i>P</i><0.05, n.s. = not statistically significant. MPO = myeloperoxidase. TMB = 3,3′,5,5′-Tetramethylbenzidine. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. ABAH = 4-aminobenzoic acid hydrazide. Activation ratio = contrast-to-noise ratio 60 minutes over 15 minutes post MPO-Gd injection.</p

Antibody capture improves the specificity of MPO activity assays on extra- and intracellular extracts in various models of inflammatory diseases.

<p>(<b>A</b>) Antibody capture of MPO followed by activity detection with ADHP reveals high specificity towards MPO. This is shown in extra- and intracellular fractions in brains from EAE mice, livers from mice with NASH, and hearts and plasma from mice with myocardial infarction (n = 3 per group). (<b>B</b>) The same samples processed without antibody capture reveal poor specificity towards MPO, and no significant difference between WT and MPO-KO mice (n = 3 per group). * <i>P</i><0.05. ** <i>P</i><0.01. *** <i>P</i><0.001. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. MPO = myeloperoxidase. EAE = experimental autoimmune encephalomyelitis. MI = myocardial infarction. NASH = non-alcoholic steatohepatitis. ECF = extracellular fraction. ICF = intracellular fraction. WT = wildtype C57BL/6. MPO^−/− = MPO knockout.</p

Spike and recovery assay: tissue homogenates and extracellular fluid contain interfering substances.

<p>(<b>A</b>) Extracellular protein fraction from different organs contains substances interfering with ADHP, luminol, and APF assays (n = 2 per group). (<b>B</b>) Intracellular protein fractions also contain interfering substances (n = 2 per group). MPO = myeloperoxidase. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. APF = 3′-(p-aminophenyl) fluorescein. HPF = 3′-(p-hydroxyphenyl) fluorescein.</p

Validation of Extracellular Protein Isolation and MPO Protein Precipitation.

<p>(<b>A</b>) LDH assay of intra- and extracellular protein fractions of different organs shows that the extracellular fraction only contains very low levels of LDH activity, while the intracellular fraction contains the majority of the LDH activity (left). LDH ratio shows a 90 or higher fold level of ICF LDH over ECF LDH activity (right). (<b>B</b>) Protein precipitation of MPO with acetone has no effect on its activity, as evaluated with ADHP (n = 2 per group). LDH = lactate dehydrogenase. BCA = bicinchoninic acid. MPO = myeloperoxidase.</p

MPO in the literature.

<p>(<b>A</b>) Usage of MPO activity assays in the Literature from 2011 to 2012. (<b>B</b>) Manuscripts published on MPO from 1990 to 2012; grey bars indicate manuscripts considered in (<b>A</b>). MPO = myeloperoxidase. TMB = 3,3′,5,5′-Tetramethylbenzidine. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. BALF = bronchoalveolar lavage fluid. Ab = antibody. APF = 3′-(p-aminophenyl) fluorescein. ELISA = enzyme-linked immunosorbent assay.</p

FMT-CT imaging of MMP and protease activity.

<p>(<b>A</b>) Representative images from sham and JVL mice show no visible signal from FMT imaging. (<b>B</b>) Quantification of the fluorescence signal demonstrates barely measurable signal and no significant difference between sham and JVL mice.</p

Histopathology of sham (top row), JVL, (middle row), and EAE (bottom row) groups.

<p>(<b>A</b>) MPO stain for MPO-positive cells/myeloid cells, (<b>B</b>) Mac-3 stain for activated macrophages/microglia, and (<b>C</b>) LFB stain for demyelination. Bar = 50 µm.</p

Weights of sham group (n = 6, open circle) and JVL group (n = 16, solid square) up to day 130 post-surgery.

<p>Weights of sham group (n = 6, open circle) and JVL group (n = 16, solid square) up to day 130 post-surgery.</p

Flow cytometric analysis of brain inflammatory cells.

<p>(<b>A</b>) Cells were pre-gated for positive CD45 expression to identify all leukocytes, and (<b>B</b>) then divided into lymphocytes, neutrophils, and myeloid cells. The total number of all leukocytes, but also lymphocytes, neutrophils, and myeloid cells in the brain was unaffected in JVL mice (n = 4) compared to sham (n = 4), but significantly increased in EAE mice (n = 5). (<b>C</b>) Differentiating macrophages/microglia from monocytes showed that there were almost no monocytes in the brain of JVL and sham mice, but both cell types were increased to high numbers in EAE mice. Lin = CD90, NK1.1, B220, CD49b, and Ly-6G.</p