Profile of infiltrating cells.

<p>A) Immunohistochemistry of PDCA1. PDCA1⁺ cells infiltrate in the cryoablated skin (▲, arrowhead). Scale bars, 20 μm. B)C) Flow cytometry analysis of infiltrating cells. B) Dots within bold square box shows PDCA1⁺B220⁺CD19^- plasmacytoid dendritic cells. Gating strategy is also shown. Dead cells, which are 7-AAD⁺, were removed. We used CD11c for mDCs, CD11b for neutrophil and macrophage, TCR γδ for γδ T cells, and CD49b for natural killer cells. Cryo, cryoablation. All data are shown as dots (C). Bars represent the mean values. Significant differences between the means are indicated, *P<0.05 versus control. n = 4, in each group.</p

Migration assay of dendritic cells to regional lymph nodes.

<p>Flow cytometry analysis for migrated FITC⁺CD11c⁺ myeloid dendritc cells and FITC⁺PDCA1⁺ plasmacytoid dendritc cells. Gating strategy is also shown. Cryo; cryoablation. All data are shown as dots. Bars represent the mean values. Significant differences between the means are indicated, *P<0.05 versus control. n = 4, in each group.</p

Functional and morphological changes of skin barrier.

<p>A) Immunohistochemistry of E-cadherin and Zo-1 after 15-sec cryoablation at day 1. Scale bar, 100 μm. The change of transepidermal water loss (TEWL, B) and corneal water content C). D) Increased skin permeability of lucifer yellow after 15-sec cryoablation at day 1. Green fluorescence shows the permeation of lucifer yellow. Cryo, cryoablation; scale bar, 100 μm; and sec, seconds. Values represent the mean ± SEM (B, C). Data are shown as dots (D). Bars represent the mean values. Significant differences between the means are indicated, *P<0.05 versus 5 sec in (B) and control in (D). n = 4, in each group of (B) and (C). n = 3, in each group of (D).</p

mRNA expression in the skin treated with cryoablation.

<p>The mRNA expressions of IFN-α1, TNF-α, and RARRES2 were measured by real time PCR analysis at day 1 in 15-sec cyoablated skin. The levels of TNF-α, and RARRES2 (potent chemoattractant of pDCs), were not increased by cryoablation. Cryo, cryoablation. All data are shown as dots. Bars represent the mean values. Significant differences between the means are indicated, *P<0.05 versus control. n = 4, in each group.</p

Cryoablation mouse model.

<p>A) Cryoablation technique using cotton swab. B) Macroscopic changes of the back after 15-sec cryoablation. C) Chronological changes of ulcer size of the back skin. The formed ulcer is larger in the 15-sec cryoablated group than in the 5-sec ablated group. D) Macrosopical changes of subcutaneous blood vessels in the 15-sec cryoablated group at day 1. E) HE stain, CD31 immunostaining (blood vessel marker), and truidine blue staining of the 15-sec cryoablated skin at day 1. Square box in HE stain shows a high power image of infiltrating cells, mainly neutrophils. The number of degranuated cells is also presented. Square box in toluidine blue stain shows high power image of degranuated mast cells (▲; arrowhead). All scale bars, 20 μm. Cryo; cryoablation. sec; seconds. HPF; high power field. Values represent the mean ± SEM (C). Data are shown as dots (E). Bars represent the mean values. Significant differences between sample means are indicated, *P<0.05 versus 5 sec in (C) and control in (E). n = 4, in each group.</p

Changes of temparature of tissues after cryoablation.

<p>A) The temperature change of skin surface after 5-sec or 15-sec cryoablation was monitored by thermography. The thermometer probe was inserted into the subcutaneous tissue. B) The temparature recovered more slowly in the 15-sec than in the 5-sec cryoablated group. The increase of temparature became blant just bellow 0°C, where recrystallization and the following tissue damage occur. sec; seconds. All values represent the mean ± SEM. n = 4, in each group.</p

Parasitaemia patterns of mice infected with FOA-resistant parasite and orally treated with FOA.

<p>The mice were treated twice daily for 3 days with FOA (40 mg/kg cumulative dose) at passages 2, 5, 10 and 12. Note that the patterns mirror each other, confirming that the acquired FOA-resistance is stable. The discontinuous curve indicates that following FOA administration to mice infected with the wild-type parasite, no parasites could be observed under the microscope until day 14 p.i when recrudescent parasites were observed.</p

Electron micrographs of FOA-resistant and wild-type parasites grown in mice in the absence of drug.

<p>In (B), the trophozoite of the wild-type parasite shows normal ultrastructural morphology with various compartments and organelles well defined by membranes that have retained their integrity. In (A), late schizont of FOA-resistant parasite is seen with intact plasmalemma housing non-viable segmenters/merozoites that appear as electron dense bodies probably due to compaction of nuclear chromatin and condensation of cytoplasm. (C) is an immuno-gold electron micrograph adapted from Bhowmick et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021251#pone.0021251-Bhowmick1" target="_blank">[114]</a> showing a similar ‘syncytial’ cell from <i>P. falciparum</i> with viable merozoites. Note the distinct nucleus of segmenters. Abbreviations: CN, condensed segmenter; E, erythrocyte; FV, food vacuole; IPV, intraparasitic vacuole; M, mitochodria; N, nucleus; NM, nuclear membrane; PM, plasma membrane; PV, parasitophorous vacuole; PVM, parasitophorous vacuolar membrane; VL, vacuolization.</p

The photomicrographs of FOA-resistant and wild-type parasites during serial passaging in the absence of drug.

<p>A and B show FOA-resistant parasite at passages 2 and 12 respectively, with dark/condensed chromatin dot & cell shrinkage characterized by small and more dense cytosol (red arrowheads), confirming that the death phenomenon was a persistent feature of the FOA-resistant parasite line. The wild-type parasite (C) shows the typical ellipsoidal morphology and full cytoplasm.</p

Resistance induces internucleosomal DNA fragmentation in FOA-resistant parasite grown in mice without drug.

<p>Polyacrylamide gel electrophoretic fraction of DNA was done in two independent experiments, A and B. In A, lanes 1 represent DNA extracted from the wild-type parasite that shows no cleavage, while lanes 2 represent cleaved DNA of FOA-resistant parasite, with the main band at ≈200 bp. Similar results are represented in B, where the wild-type shows no cleaved DNA (lanes 1), while FOA-resistant parasite shows cleaved DNA and a clear band at ≈200 bp (lanes 2). Lane M is the molecular size marker (pSG5/Hinf I and φX174/HincII for A, and pSG5/Hinf I for B).</p