第758回千葉医学会例会・第9回千葉大学第三内科懇話会 25.

HCV prevalence. (TIF 566 kb

Elution of DSS tested in anti-HTLV-1/-2 ELISA.

<p>Serial dilutions of sera from six HTLV-1 infected individuals (H1_#1 –H1_#6), eight HTLV-2 infected individuals (H2_#1 –H2_#8) and one healthy donor (HD) were serially diluted and spotted on filter paper in duplicate (grey = eluate filter #1, hatched = eluate filter #2). Samples were eluted (resulting in an additional 1:15 dilution) and tested in the commercial Murex HTLV-1/-2 ELISA. The cut-off (dashed line) was calculated as the mean OD of three replicates of the negative control normal human sera plus 0.2, according to the manufacturer's instructions.</p

Sera tested in anti-HTLV-1/-2 ELISA.

<p>Serial dilutions of sera from six known HTLV-1 infected individuals (H1_#1 –H1_#6), eight HTLV-2 infected individuals (H2_#1 –H2_#8) and one healthy donor (HD) were tested with the commercial Murex HTLV-1/-2 ELISA. The cut-off (dashed line) was calculated as the mean OD of three replicates of the negative control normal human sera plus 0.2, according to the manufacturer's instructions.</p

ELISA and confirmatory Western blot results of the validation.

<p>ELISA and confirmatory Western blot results of the validation.</p

Human BPI-peptide damages the IAV particles.

<p>Virus particles were incubated either with 100 (100) and 500 μg/mL (500) of human (A) or murine BPI-peptides (B) for 1 h or left untreated (C). After the incubation the virus particles were visualized by transmission electron microscopy. Therefore, the particles were negatively stained with 2% uranylacetate and transmission electron microscopy was carried out using a JEOL TEM 2100 at 120kV. Micrographs were recorded with a fast-scan 2k x 2k CCD camera F214. One representative experiment out of 3 performed is displayed.</p

BPI peptides used.

<p>BPI peptides used.</p

Human BPI-peptide inhibits the activation of PBMCs by Influenza A virus.

<p>Human PBMCs were isolated by dense gradient centrifugation from buffy coats. Thereafter, the cells were seeded and stimulated with purified Influenza A virus (MOI 2) (H1N1) in the presence (black bars) of increasing concentrations and absence (white bar) of human and mouse BPI-peptides (50 μg/mL, (grey bar)), and left unstimulated (control). In addition, the cells were incubated in the presence of either human BPI-peptide (50 μg/mL, huBPI) or mouse BPI-peptide (50 μg/mL, mBPI), respectively. After 20 h the supernatant was collected and the release of IFNα (A) as well as IL-6 (B) was determined by a specific ELISA. One representative experiment out of three is displayed. Statistically significant differences are given as p values (* <0.05 and ** <0.01); n = 3 ± SEM.</p

Human BPI-peptide specifically inhibits the replication of different IAV strains.

<p>Protease-deficient MDCK(H) cells were infected with 500 PFU/well of Influenza A virus strain A/PR/8/34 (H1N1) (A) and strain A/Aichi/2/68 (H3N2) (B) for 1 h. After the infection the virus containing supernatant was removed and the cells were grown for additional 13 h. Thereafter, the fixed and permeabilized cells were incubated with the mouse anti–nucleoprotein IAV monoclonal antibody. The binding of the antibody was detected by a donkey anti-mouse IgG-HRP antiserum and adding the reagent TMB Super Sensitive One Component HRP Microwell Substrate. Substrate conversion was detected by 450 nm. For the control sample is n = 8 ± SEM, all other samples n = 5 ± SEM. C) Calu-3 cells were infected with 300 PFU/well of Influenza A virus strain A/Aichi/2/68 (H3N2). Prior to the infection the virus was incubated in the presence or absences of the indicated amount of human or mouse BPI-peptide for 1 h. Thereafter, the virus solution was removed and the cells were incubated for 24 h. The virus amount in the supernatant was analysed by adding an aliquot of the supernatant to MDCK (H) cells for 1 h. After the infection the virus containing supernatant was removed and the cells were grown for additional 13 h. Thereafter, the fixed and permeabilized cells were incubated with the mouse anti–nucleoprotein IAV monoclonal antibody and detected as outlined above. (D) Furthermore, also the release of CCL5 into the supernatant of the infected Calu-3 cells was determined via a specific ELISA. One representative experiment out of 5 performed is displayed. Statistically significant differences are given as p values (** <0.01 and *** <0.001); n.s. is not significant; n = 3 ± SEM.</p

Mutant human BPI-peptide corresponding to the mouse peptide loose its activity and human BPI peptide inhibits the replication of VSV.

<p>BHK-cells were infected with 360 PFU/well of VSV-GFP virus. Prior to infection the VSV was incubated with 20 μg/mL of human BPI (huBPI) and mouse BPI-peptide (mBPI), respectively or remained untreated (control) for 1 h. After 1.5 h of the cells were overlaid with 1.25% Avicel medium and incubated for 42 h. Thereafter, the cells were fixed and stained with crystal violet for 1 h at 4°C. Finally the plaques were counted (A). MDCK(H) cells were infected with 500 PFU/well Influenza A virus strain A/PR/8/34 (H1N1) for 1 h. Before the infection the virus was incubated in the presence or absence of 20 μg/mL of either human BPI-peptide (huBPI), murine BPI-peptide (mBPI), human BPI-peptide ^N1^D (mutant 1), human BPI-peptide ^K11^M (mutant 2), human BPI-peptide ^N1^D and ^K11^M (mutant 3) or left untreated (control) for 1 h (B) or incubated in the presence or absence of 20 μg/mL of either human BPI-peptide (huBPI), murine BPI-peptide (mBPI), mouse BPI-peptide ^D1^N and ^M11^K (mutant 4) or left untreated (control) for 1 h (C). After the infection the virus containing supernatant was removed and the cells were grown for additional 13 h. Thereafter, the fixed and permeabilized cells were incubated with the mouse anti–nucleoprotein IAV monoclonal antibody. The binding of the antibody was detected by a donkey anti-mouse IgG-HRP antiserum and adding of the reagent TMB Super Sensitive One Component HRP Microwell Substrate. Substrate conversion was detected by 450 nm. One representative experiment out of 3 performed is shown. Statistically significant differences are given as p values (*** <0.001); n.s. is not significant; n = 5 ± SEM.</p

Lack of infection of XMRV susceptible LNCaP cells by co-culture with activated PBMCs.

<p>PCR results with isolated LNCaP cell DNA after co-culture with PBMCs from CFS patients (lane 1–5) and healthy donors (lanes 6–10). Five representative samples out of 10 co-cultures for each group are shown. As control, LNCaP cells were infected with XMRV-containing supernatant from 22Rv1 cells (lane 12). A water-only control (no template control, NTC) was run in lane 11. Results of the GAPDH PCR with the same samples are shown in the lower panel. M  = 100 bp marker.</p