Altered PrP expression pattern in CSF of CJD patients.

<p>CSF samples from CJD patients (N = 40) and non-CJD controls (N = 16), were obtained by lumbar puncture. <b>A.</b> PrP levels in CSF were examined by Western blot. <b>B.</b> and <b>C.</b> Quantification of relative levels of total PrP protein in CSF and the ratio of the PrP band 1 (upper) and PrP band 2 (lower) in CSF, respectively. <b>D.</b> Comparison between ratios of PrP band 1 and 2 in sCJD versus fCJD <b>E.</b> Correlation of total PrP protein levels with 14-3-3 protein in each CSF samples presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036159#pone-0036159-g001" target="_blank">Fig 1</a>. In B, C and D the red line represents the mean of all samples analyzed. Statistical significance was calculated using Student's t-test.</p

Low correlation between 14-3-3 protein levels and LDH activity in CSF of CJD patients.

<p>CSF samples from CJD patients (N = 40) and non-CJD controls (N = 16), were obtained by lumbar puncture. Then, <b>A.</b> 14-3-3 levels in CSF were analyzed by Western blot. <b>B.</b> Relative levels of 14-3-3 protein band were quantified using densitometric analysis. <b>C.</b> LDH activity was measured in each sample as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036159#s4" target="_blank">Material and Methods</a>. <b>D.</b> A correlation between 14-3-3 protein and LDH activity from panels B and C is presented. <b>E.</b> Quantitative analysis of total protein levels in CSF samples of panel A was performed. In B, C and E the red line represents the mean of all samples analyzed. Statistical significance was calculated using Student's t-test.</p

Alterations in PrP expression pattern in CSF are specific to CJD patients.

<p>CSF samples from healthy control subjects (N = 3), CJD patients (N = 5), dementia patients (N = 3) and paraparesis patients (N = 3), were obtained by lumbar puncture. <b>A.</b> Assessment of PrP levels (two upper panels with different exposure), and 14-3-3 protein levels (third panel) by Western blot analysis, and total protein content by Ponceau Red staining (lower panel, a selected band is presented). <b>B.</b> and <b>C.</b> Quantification of relative total PrP protein levels in CSF and the ratio of the PrP band 1 (upper) and PrP band 2 (lower) in CSF, respectively. The asterisk in A indicates one sample with a technical problem in the blot detection that was excluded for quantification of panel B and C. In B and C the red line represents the mean of all samples analyzed. Statistical significance was calculated using Student's t-test.</p

Changes in the glycosylation pattern of PrP in CSF of CJD patients.

<p><b>A.</b> The pattern of expression of PrP in two representative CSF samples (from one healthy control individual and one CJD patient) is compared to the PrP pattern observed in control brain tissue using Western blot analysis. <b>B.</b> CSF samples from one healthy control individual, one CJD patient and one healthy control brain tissue were treated with PNGase F and then analyzed by Western blot. Data represent the analysis of three control and five individual CJD samples. <b>C.</b> and <b>D.</b> CSF samples from one healthy control individual and one CJD patient were treated with various concentrations of PK or thermolysin (Ther), respectively, followed by Western blot analysis. Data represent the analysis of three control and five individual CJD samples.</p

Changes in 14-3-3 protein and PrP levels in CSF from six CJD patients overtime.

<p>PrP and 14-3-3 protein levels were analyzed in CSF samples obtained by lumbar puncture of 6 CJD patients (CJD-1 to CJD-6) at different time points during disease. Total protein content was assessed by Ponceau Red staining, the predominant band (albumin) is shown. The asterisk in samples from CJD-2, CJD-5 and CJD-6 indicates an additional PrP protein band detected between PrP band 1 and band 2.</p

Alteration of BBB functions in the infected hCMEC/D3 cells.

<p>(A) Altered permeability of a monolayer of infected hCMEC/D3 cells. The endothelial cells were cocultured with irradiated MT-2 or C81-66 lymphocytes for 15 days. Then hCMEC/D3 cells were seeded on Transwell filters and permeability to FITC-dextran 70 kDa was assessed after differentiation of the monolayer. (B) Effect of endothelial cells infection on CEM lymphocyte migration. Infected endothelial cells were seeded onto filters. The migration was estimated at 24 hours of culture by fluorescence assay after labeling of lymphocytes (from the CEM, Jurkat, MT-2 of C81-66 cell-lines) with a fluorescent marker. The migration rate is expressed as ratio (%) of fluorescence intensity in the lower compartment versus total fluorescence. (C) Analysis of the expression level of ZO-1, Occludin, and viral p24 of a monolayer of hCMEC/D3 cells cocultured with irradiated MT-2 or C81-66 for 15 days. β-tubulin was used for normalization. (D) Inhibiting MLC phosphorylation has no effect on the permeability for FITC dextran 70 kDa across a monolayer of infected endothelial cells. hCMEC/D3 cells were seeded on filters. After differentiation, cultures were either left untreated of treated for 48 hours with ML-1, a specific inhibitor for MLCK activity. Permeability was then estimated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000205#s4" target="_blank">Materials and Methods</a>.</p

Detection of Glut-1 and HTLV-1 transcripts in the thoracic spinal cord from HAM/TSP patient.

<p>(A,B,C) Glut-1 staining by iPO (DAB substrate, brown) on thoracic spinal cord sections from a HAM/TSP patient. Nuclei were counterstained with Harri's haematoxilin solution (blue). (A,B) Cryostat section; (C) paraffin section. Magnification: (A) 30×; (B,C) 75×. (D,E) Detection of HTLV-1 transcripts (tax) in cryosections of the spinal cord by <i>in situ</i> hybridization. Arrow heads indicate positive cells and vascular structures. Astrocytes were detected by iPO (DAB substrate, brown) against GFAP. Magnification: 220×.</p

Productive infection of hCMEC/D3 cells by HTLV-1.

<p>(A) Kinetics of p19 viral protein secretion in the supernatant of hCMEC/D3 and irradiated HTLV-1 MT-2 lymphocyte cocultures. Cells were cultivated or not in presence of 25 µM AZT. Results are mean and standard deviation from triplicate experiments. (B) Kinetics of detection of infected hCMEC/D3 cells by irradiated MT-2 cells. Infection was assessed by FACS analysis of p24 viral protein at days 12, 14, 16 and 22 post-coculture. (C) Production of infectious viral particles by HTLV-1-infected endothelial cells using a reporter cell-line (293T-LTR-GFP). The supernatant of hCMEC/D3 infected cells was collected and ultracentrifuged and the resuspended pellet was applied on the reporter cell-line 293T-LTR-GFP. The expression of GFP was assessed 6 days later after cell fixation.</p

Expression of HTLV-1 receptors, Glut-1 and NP-1, in the thoracic spinal cord from an uninfected individual.

<p>(A,B) Glut-1 immunostaining by immunoperoxydase technique (iPO) (DAB substrate). (C) Dual staining for Glut-1 (iPO, DAB substrate, brown color) and factor VIII (Alcaline phosphatase (AP), FastBlue substrate, blue color). (D,E) NP-1 staining by iPO technique (DAB substrate). Thoracic spinal cord specimens were cut in a cryostat, fixed in methanol and processed for iPO as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000205#s4" target="_blank">Materials and Methods</a>. (F) Dual staining for NP-1 (iPO, DAB substrate, brown color) and factor VIII (Alcaline phosphatase, FastBlue substrate, blue color). Frozen tissue samples from the thoracic spinal cord were cut on a cryostat at 10 µm, fixed in methanol and processed for immunohistochemistry as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000205#s4" target="_blank">Materials and Methods</a>. Magnification: (A,D) 5×, (B–E) 50×, (C–F) 100×.</p

Syncytia formation between hCMEC/D3 cells and HTLV-1 infected MT-2 lymphocytes at 24 h post-contact.

<p>(A) Role of the viral proteins in the formation of the syncytia. Evaluation of the number and size (nb of nuclei/syncytium) of the syncytia obtained by coculture between hCMEC/D3 and MT-2 cells in the presence of serum from a HAM/TSP patient (1/7) (noted HAM/TSP) or from an uninfected individual (control, noted HTLV negative control or in the absence of any serum (w/o serum). (B) Role of the viral receptors in the formation of the syncytia. Evaluation of the number and size (nb of nuclei/syncytium) of the syncytia obtained by coculture between hCMEC/D3 and MT-2 cells in the presence of dextran sulfate in blue (that prevents the HTLV-1 Env/HSPG interaction), or in the presence of VEGF165 in green (that prevents the HTLV-1 Env/NRP-1 interaction), or in the presence of a polyclonal antibody against Glut-1 in red (that prevents the HTLV-1 Env/GLUT1 interaction). Results are representative of 3 independent experiments. (C) Detection of viral p24 protein (green) by immunofluorescence in a syncytium. Nuclei were stained with DAPI (blue). (D) Demonstration of endothelial origin of the syncytia by prior labeling of hCMEC/D3 cells with a red vital fluorescent marker (Cell-tracker, red). Nuclei were stained with DAPI (blue). Magnification (B–C): 350×.</p