Surface markers of microparticle.

<p>BAL was double stained with annexin V and cell surface markers and analyzed by a flow cytometer: isotype control (A), CD11b (B), CD41a (C), CD45 (D), keratan sulfate (E), and SPD (F). MPs were gated using 1.33 μm beads as size markers, and the annexin V-positive gate was analyzed for the expression of the surface markers. To distinguish MPs from apoptotic bodies, BAL was double stained with annexin V and PI (G) using apoptotic bodies from hydrogen peroxide treated HT29 cells as a positive control (H). To detect surface sialic acid on MPs, BAL was double stained with annexin V and lectins. Annexin V-positive gate was analyzed for the expression of SNA (I) and MAA1 (J). The histrogram are representative of ten BAL samples.</p

Anti-viral activity by HI against H3N2 influenza A virus of BAL MP preparations with and without the sialidase treatment.

<p>(A) Pooled and purified BAL MP was mixed with PBS or RDE at a 1:3 ratio. The samples were rotated at 4°C overnight followed by sialidase inactivation at 56°C for 30 minutes. A sham treated sample was also tested in parallel. The data were derived from 6 pooled BAL samples run in duplicate. The HI titers are shown as the geometric mean±SEM and compared by a <i>t</i>-test. The three asterisks (***) represent a significant p-value < 0.0001 by ANOVA. (B) The sialidase treatment did not permanently inactivate the viral infectivity. A/Thailand/Siriraj-04/2003 (H3N2) virus was incubated with either MP or media at 37°C for 1 hour. RDE at a 1:2 ratio was added into either the virus only or the MP+virus and incubated at 4°C overnight. After the incubation, the virus was inoculated into MDCK cells and the viral NP protein was measured after an overnight incubation. The data represented a triplicate result for each condition. The two asterisks (**) represent a significant p-value < 0.01 by the <i>t</i>-test.</p

Transmission electron microscopy of the high-speed-centrifuged BAL pellet mixed with the viral preparation.

<p>(A) Binding of MP with the smooth surface (narrow arrow) and influenza virion with spikes (A/Thailand/104/2009) (wide arrow); (B) binding of MP (narrow arrow) with multiple virions (wide arrow); and (C) free exosome and MP with smooth membrane appearance shown with narrow and wide black arrows, respectively. The exosome size was usually smaller than 100 nm, whereas the MP was 100–1000 nm in diameter.</p

BAL fractionation using FPLC with a Hiprep sephacryl S-500 16/60 column and anti-viral activity in each BAL fraction.

<p>(A) The UV (280 nm) absorbance peak as an indicator of the protein concentration of the virus (A/Thailand/MVCU-013/2009) preparation. (B) The UV absorbance peaks of one BAL sample (upper) together with HI titers against pdmH1N1 (A/Thailand/104/2009) from 2 BAL samples (2 sets of fractions) (lower). The HI assay was run in duplicate and repeated by another set of BAL fractions. Each point of the HI titers is shown as the mean±SEM. The molecular mass at the peak of the HI titers was estimated from a dextran molecular weight standard.</p