Relative resistance of PESSc prions to ProteinaseK.

<p><b>A)</b> Cell lysates from PESSc cells, ScN2a cells and SMBRC040 cells were each divided into 13 aliquots. All aliquots, but one of each cell line, were subjected to ProteinaseK digestion (25 µg/ml; at 37°C). Every two hours the digestion of one aliquot per cell line was stopped with PMSF. PrP^res was detected by dot-blot analysis (mAB ICSM18). In both murine cell lysates PrP^res was detectable for up to 22 hours; in PESSc cell lysates PrP^res was still detectable after 24 hours. <b>B)</b> Cell lysates from uninfected PES cells, PESSc cells and ScN2a cells were divided into six aliquots each, ProteinaseK was added at increasing concentrations (0 µg/ml, 25 µg/ml, 100 µg/ml, 250 µg/ml, 500 µg/ml and 1000 µg/ml), and the samples were incubated for one hour at 37°C. The digestion was terminated by adding PMSF. PrP^res was detected by dot-blot analysis (mAB ICSM18). While 25 µg ProteinaseK/ml were sufficient to clear the control lysate from any detectable PrP, strong PrP^res signals were detected in both prion infected samples up to 500 & 1000 µg ProteinaseK/ml.</p

Re-infection of „cured“ PESSc cells with different sheep scrapie field isolate.

<p>PESSc cell clones A2/A5 and E6/C5, propagating ovine scrapie prions from the sheep scrapie field isolate S71/04, had been cured with the prion inhibitors Imatinib, Suramin and Pentosanpolysulfat until any detectable PrP^res was cleared from the culture. Following that, the cells were cultured for 18 to 22 splits in the absence of the inhibitors and remained uninfected as monitored by dot-blot analysis. The cells were then subjected to infection with a second ovine scrapie field isolate, S95/04, and split 20 times at a ratio of 1:2. PrP^res was detected by dot-blot analysis. mAB: ICSM18.</p

Selected cell repository test cell lines.

<p>^#<u><b>bold</b></u>: successful infection; <i>italic</i>: transient infection, but not repeatable (data not shown)</p><p>Selected cell repository test cell lines.</p

Persistent infection of a bovine cell line (PES) with sheep scrapie prions.

<p><b>A)</b> PES cells were infected with the natural sheep scrapie isolates S71/04 and S95/04. Both isolates were derived from sheep carrying the ARQ/ARQ prion genotype. PrP^res was detected by dot-blot analysis. Shown here are samples from three independent infections with S71/04, assayed 25, 110 or 45 cell passages after inoculation. S95/04-infected PESSc cells were used for cell cloning and shown here is PESSc clone G6. Uninfected control cells (ᴓ) or PES cells challenged with BSE or RML prions that were passaged 28 and 21 times, respectively, show no detectable PrP^res signal. In addition the challenge with four other scrapie field isolates (S13/04, S120/04, S90/04 and S66/04) did not lead to PrP^res propagation; shown here are samples after 10 cell passages post inocula application. <b>B)</b> Serial cloning of PESSc cells that had been infected with scrapie isolate S71/04 resulted in cell populations with different PrP^res loads. Cell clones with stronger PrP^res signals were selected for further cultivation. <b>C)</b> Detecting PrP^res by western-blot PESSc prions were recognized by a panel of four different antibodies (ICSM18, 6H4, L42, P4) and show a higher molecular weight compared with BSE, scrapie and RML brain homogenate. <b>D)</b> The Cell-ELISA confirmed the infection of PES cells, showing PrP^res positive single cells and cell accumulations. Uninfected PES cells are shown in the inlet.</p

Synthetic oligonucleotides used for RT- PCR and QPCR

<p>ACTNB, β-actin; ATP7A, ATPase 7A; CDH1; E-cadherin; CLDN, claudin; DMT1, divalent metal transporter-1; LEPR, leptin receptor; OCLN, Occludin; TFRC, transferrin receptor; ZnT1, zinc transporter-1; ZO-1, tight junction protein ZO-1.</p

PCP-R displayed continuous tight junction strands.

<p>The cells, grown on coverslips, were stained for detection of ZO1 (A), Occludin (B) and Claudin-1 (C). Secondary antibodies were labeled with Alexa Fluor 594 (Alexa-594, red). Pictures presented are Apotome-generated images; <i>bottom</i> of each panel is an <i>xy</i> en face view of a cell culture monolayer shown in a maximum-intensity projection through the z-axis; <i>top and side</i> of each panel is a cross section through the z-plane of multiple optical slices. The basolateral side of PCP-R is oriented towards the top or right side, respectively, of the top and right images of each panel. In (A) and (B) the actin cytoskeleton was in parallel stained with phalloidin Alexa Fluor 488 (green). Since Claudin-1 samples were fixed with methanol we could not observe a qualitatively sufficient actin staining. In all samples nuclei were stained with DAPI (blue). The images shown are representative examples of multiple stainings.</p

Comparison of mRNA expression levels of genes encoding for transporter proteins and junctional proteins in PCP-R and porcine CP tissue.

<p>The mRNA expression of all analyzed genes was normalized that of β-actin in the same sample. The expression in porcine CP tissue was arbitrarily set as 100%. Data represent mean ± SD (n = 3).</p>*<p>p<0.05.</p>**<p>p<0.01.</p>***<p>p<0.001 as compared to controls.</p

Generation of a stable subcultivatable porcine choroid plexus cell line.

<p>(A) Light microscopic depiction of primary porcine choroid plexus epithelial cells. (B) Subculture 43 (day 4): pure population of epithelial choroid plexus cells. These cells were continuously subcultivatable. (C) Immunocytochemistry with a mouse-anti human cytokeratin monoclonal antibody. PCP-R express the epithelial cell marker cytokeratin. (D) Immunofluorescence analysis of PCP-R shows expression of cytokeratin (green). The nuclei are counterstained with propidium iodide (red). Subculture 40 was analyzed in (C) and (D).</p

Scheme for treatment of the cell cultures with Ara-C

<p>Ara-C, cytosine arabinoside.</p

PCP-R display barrier functions when grown on cell culture inserts (high TEER values correlate with low FITC-inulin flux through PCP-R-layers).

<p>1.5×10⁴ (white bars) or 2.5×10⁴ (grey bars) PCP-R cells were seeded on cell culture inserts; TEER values (A) and permeability coefficients for FITC-inulin (B) were measured over time. Shown is the mean ± SD of at least three experiments performed in duplicates.</p