Comparison of the histopathological changes after KA/E2 treatment in WT and TLR2 KO mice.

Tissue inflammation was observed in the images of lung sections after H&E or PAS staining (A). Representative inflammation scoring and PAS-positive cells (mean ± SEM). A grade of 0 indicates no detectable inflammation and a grade 4 indicates high percentages of airways and blood vessels in section cuffing by inflammatory cells (0 = normal tissue; 1 = 75%). Severity scoring was based on the thickness of the bronchi or vessels surrounding inflammatory cells (0 = no cells; 1 = 1–3; 2 = 4–6; 3 = 7–9 cells thick; 4 = 10 or more cells thick). Means of the p-value were calculated for comparison to the control (n = 5 mice/group, 3 independent experiments, *; p < 0.05).</p

Comparison of DC surface marker activation and the production of Th2-related cytokine expressing cells.

Activated BMDCs surface marker (A) and IL-4 expressing T cells (B) were compared between the WT and TLR2 KO groups. (n = 3/group, 3 independent experiments, *; p p p < 0.001).</p

KA/E2 induced allergic airway inflammation via TLR2.

Lung inflammation was induced via intranasal inoculation of 4x105 KA/E2 trophozoites (A). Airway resistance values in response to methacholine (0 to 50 mg/ml) were compared between WT and TLR2 KO mice treated with KA/E2 (B). Differential inflammatory cells were counted in BAL from WT or TLR2 KO mice using a microscope (C). (n = 5 mice/group, 3 independent experiments, *; p < 0.05).</p

Evaluation of the inflammatory-related gene expression owing to KA/E2 ES-P in primary lung cells from WT and TLR2 KO mice.

Assessment of the expression of inflammatory response related genes in primary lung cells from WT and TLR2 KO mice. (n = 3/group, 3 independent experiments, *; p p p < 0.001).</p

Comparison of the Th2 cytokine expression level after treatment with KA/E2 in WT and TLR2 KO mice.

Th2 related cytokine (IL-4, IL-5, and IL-13) concentrations in the BALF (A) and culture supernatants of CD3-stimulated lymphocytes from LLN (B) were determined using ELISA. The plates were read at 450 nm on a standard ELISA reader, VICTOR 3 (Each p-value was determined using t-test methods compared to the control) (n = 5 mice/group, 3 independent experiments, *p p < 0.01).</p

KA/E2 ES-P induced inflammatory-related gene expression by TLR2 in MLE12 cells.

Comparison of the expression of inflammatory response-related genes in MLE 12 cells from the KA/E2 ES-P group or TLRs antagonist pre-treated group. (n = 3/group, 3 independent experiments, *; p p p < 0.001).</p

Cell membrane Permeability changes, mitochondrial membrane potential changes, and the effect of <i>Torreya nucifera</i> on ATP production in <i>A</i>. <i>lugdunensis</i>.

Unlike the positive control group (Triton X-100), which showed high fluorescence, the groups treated with 25 μg/mL and 50 μg/mL of T. nucifera showed significantly low fluorescence similar to that seen in the negative control group (A). In the control group, the mitochondrial membrane potential gradient was normal (B). Groups treated with 25 μg/mL (C) and 50 μg/mL (D) of T. nucifera showed the collapse of the mitochondrial membrane potential gradient indicated by the green fluorescence in the cytoplasm. The scale bar represents 20 μm. ATP production showed that the luminescence amount was significantly reduced in the groups treated with 25 μg/mL and 50 μg/mL of T. nucifera compared to that in the control group (E).</p

Model diagram of the cellular biological mechanism of the amoebicidal effect of <i>Torreya nucifera</i>.

When A. lugdunensis were treated with T. nucifera, the morphological change of encystation was confirmed. The decrease in mitochondrial ATP level and collapse of the mitochondrial membrane potential is the likely amoebicidal mechanism of T. nucifera.</p

Image-based cytometer analysis to determine cell death after 24 hours of treatment with <i>Torreya nucifera</i>.

The process of cell death was evaluated using the Tali™ Image-based Cytometer using the Tali™ apoptosis kit. In the groups treated with 25 μg/mL (B) and 50 μg/mL (C) of T. nucifera, the green and red fluorescence indicating apoptosis and necrosis, respectively were clearly observed unlike that in the control group (A). The scale bar represents 20 μm.</p

Morphological changes and cell viability in <i>A</i>. <i>lugdunensis</i> after 24 hours of treatment with <i>Torreya nucifera</i>.

Compared with the control group (A, D, G), groups treated with 25 μg/mL (B, E, H) and 50 μg/mL (C, F, I) of T. nucifera showed morphological changes in which encystation from trophozoites to cysts and shrunken and unviable cells were observed. Cell viability was significantly decreased in the T. nucifera treated group and was dose-dependent (J). The scale bar represents 50 μm..</p