Effect of tetracyclines following peripheral scrapie infection.

<p>PrP^Sc immunohistochemistry of brain tissue (A), spinal cord (B), spleen (C), and Peyer's patches (D) of hamsters at the terminal stage of disease after intramuscular inoculation of 263K scrapie infected brain homogenate alone at 10⁻⁴ dilution. All samples were counterstained with hematoxylin. The pictures are representative of the immunohistochemical results for all terminal animals. Magnification scale bar: 1 mm (A), 250 µm (B-C-D). (E) Survival of hamsters injected intramuscularly with a 10⁻⁴ dilution of 263K scrapie-infected brain homogenate followed one hour later by a single intramuscular dose of doxycycline (10 mg/kg) at the same site. Untreated animals (□), Doxycycline (•). (F) Survival of hamsters injected intramuscularly with a 10⁻⁴ dilution of 263K scrapie-infected brain homogenate followed one hour later by an intraperitoneal dose of 10 mg/kg of tetracycline, doxycycline or minocycline, then every 2 days up to 40 days post-infection. Untreated animals (□), Tetracycline (▵), Doxycycline, (•), Minocycline (○). (G) Survival of hamsters injected subcutaneously with a 10⁻⁴ dilution of 263K scrapie-infected brain homogenate followed four days later by an intraperitoneal dose of 10 mg/kg of doxycycline, then every 2 days up to 44 days post-infection. Untreated animals (□), Doxycycline, (•).</p

Tetracycline treatment schedules and survival of hamsters infected with 263K scrapie strain.

<p>Liposomes containing doxycycline or minocycline (LipoDoxycycline and LipoMinocycline) were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001888#s4" target="_blank">Methods</a>.</p>*<p>At this time the clinical symptoms of disease appeared in 50% of animals.</p>**<p>In brackets the 95% confidence interval of the hazard ratio.</p

Effect of tetracyclines following intracerebral scrapie infection.

<p>(A) Immunoblot analysis of 263K scrapie infected brain homogenate (1%) after incubation in the absence (lane 1), or presence of doxycycline (lanes 2, 3), liposome-containing doxycycline (lane 4) and empty liposomes (lane 5), followed by proteinase K digestion. The blots were probed with the antibody 3F4 (1∶5,000). Molecular mass markers are indicated to the left. (B) Cerebral cortex of hamster four days after an intracerebroventricular infusion of 25 µg/20 µl liposome-containing doxycycline. Doxycycline-related fluorescence appears to be partly diffused in the neuropil and partly associated with nerve cell bodies and processes (arrows) and glial cells. Magnification scale bar: 50 µm. (C) Survival of hamsters injected intracerebrally with a 10⁻⁴ dilution of 263K scrapie-infected brain homogenate followed 60 days later by a single intracerebroventricular infusion of 25 µg/20 µl doxycycline or minocycline-containing liposomes. By this time 50% of infected animals showed initial clinical symptoms of disease, i.e. hyperreactivity to tactile and acoustic stimulations. Untreated animals (□), LipoDoxycycline, (•), LipoMinocycline (○).</p

Neuropsychological testing results from the 10 cases presented.

<p>All tests scores are corrected referring to respective Italian normative data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120197#sec008" target="_blank">methods</a>).</p><p>*: These are raw scores, as it was not possible to correct scores for Years of Education and Age.</p><p>**: These scores refer to a modified version of the PPT.</p><p>§: This patient underwent similar naming and comprehension tests from a different battery (BADA); see text for details.</p><p>°: Numeric scores were not available.</p><p><b>Bold scores</b> are pathological.</p><p>Neuropsychological testing results from the 10 cases presented.</p

Demographic summary of the studied cohort.

<p>Statistics are indicated as follows: Mean ±SD (RANGE)</p><p>L>R: Left Hemisphere hypometabolism Asymmetry</p><p>R>L: Right Hemisphere hypometabolism Asymmetry</p><p>Demographic summary of the studied cohort.</p

¹⁸F-FDG-PET analyses of our cohort.

<p><b>A)</b> Single-subject ¹⁸F-FDG-PET, SPM-t maps (FWE p<0.05) overimposed on a standard MNI template. Axial View. Right side showing hemispheric asymmetry. <b>B)</b> Commonalities analyses, inflated view (uncorrected p<0.0001). Dark gray areas represent sulci, light gray areas represent gyri. <b>C)</b> ROIs showing significant negative correlation between hypometabolism and performance in confrontation naming neuropsychological task (uncorrected p<0.05). See text for details. ROIs were visualized with BrainNet Viewer (<a href="http://www.nitrc.org/projects/bnv/" target="_blank">http://www.nitrc.org/projects/bnv/</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120197#pone.0120197.ref104" target="_blank">104</a>].</p

PK digestion assay of amyloid #4, #19, #28 and #32.

<p>Western blotting of PK digestion assay (amyloids #4, #19, #28, #32) showed partial protease K (PK) resistance of recMoPrP(23–231) (amyloids #4 and #28). RecMoPrP(23–231) amyloids (PK- lanes) were digested with PK at ratio 1:10 (w/w) (PK+ lanes) and 1:1 (w/w) (PK++ lanes). Western blots were performed using Fab D18 monoclonal antibody (1μg/mL). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences)</p

PK digestion assay of GT1 and N2a cells collected at different passages after infection with amyloid fibrils.

<p>Western blotting of GT1 and N2a cell lines infected with PrP amyloid #4 was observed throughout, from first passage (P1) to sixth passage (P6) and after treatment with proteinase K at ratio 1:500 (w/w). Western blot was performed using Fab D18 monoclonal antibody (1μg/mL). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences).</p

Seeding assay of N2a and GT1 cell cultures with synthetic amyloid fibrils.

<p>Seeding of recMoPrP(23–231) amyloid preparations induced the conversion of endogenous PrP^C to mildly PK resistant forms (A) and accumulation (B) in mouse neuroblastoma N2a and mouse hypothalamic GT1 amyloid-infected cell lines analyzed six passages after the infection (P6). Western blotting shows the partial protease K (PK) resistance of N2a and GT1 amyloid fibril-infected cell lysates. Fibril-infected cell lysates (PK-lanes) were digested with PK at ratio 1:500 (w/w) (PK+ lanes). Western blots were performed using Fab D18 monoclonal antibody (1μg/mL) for GT1 infected cells and Clone-P (1μg/mL) for N2a infected cells. Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences) (A). Immunofluorescence imaging shows the accumulations of PrP in N2a and GT1 amyloid fibril-infected cell lines. The deposition and level of PrP (green) in amyloid fibril-infected cell lines after six passages were detected by Fab D18 monoclonal antibody (10 μg/mL final concentration). The nuclei (blue) were stained with DAPI. Scale bar is 20μm (B).</p

PMCA analysis of raw fibrils and cell lysates from infected N2a and GT1 cell lines.

<p>Seeding ability of amyloid #4 and #28 by means of PMCA using brain homogenates of CD1 mice as substrates for amplification (A). PMCA analysis of GT1 and N2a cell lysates (infected with preparations #4 and #28) and collected at passage six (P6) after the infection (B). Black arrow indicates PK-resistant PrP. Asterisk indicates unspecific binding. Western blots were performed using 6D11 monoclonal antibody to PrP (0.2 μg/mL, Covance). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized using a G:BOX Chemi Syngene System.</p