17 research outputs found
MSU crystals inhibit protein synthesis at concentrations that induce processing and release of IL-1ß.
<p>WT BMDM were primed with LPS for 4 h prior to exposure MSU or MPU at indicated concentrations. A) BMDM in triplicate wells were pulse-labeled in medium containing [<sup>3</sup>H]-leucine for 15 min prior to harvest at the indicated times and the amount of [<sup>3</sup>H]-leucine incorporation was measured. B) Cells were harvested 4 h after addition of indicated concentrations of MSU and MPU. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting. P38 MAPK was loading control.</p
Inhibition of protein synthesis by dsRNA and inhibition of IL-1ß processing by MG-132.
<p>A) BMDM were treated with or without 4 h of LPS priming, as indicated. Cells were then rinsed in fresh medium and treated with either LipofectAMINE 2000 or LipofectAMINE 2000-poly I:C complex for 4 h, in the presence or absence of 30 µM MG-132, as indicated. Cell lysates (cell) or media (medium) samples were subjected to immunoblotting with the antibodies indicated. B) BMDM were treated with either LipofectAMINE 2000 alone or with LipofectAMINE 2000-dsRNA complex for the times indicated. Fifteen minutes before each time-point, 1 µCi of [<sup>3</sup>H]-leucine was added, and leucine incorporation was terminated by trichloroacetic acid. Each treatment was conducted in triplicate wells, and values are shown as mean ± S.D. Percent incorporation of [<sup>3</sup>H]-leucine at each point was calculated as the [<sup>3</sup>H]-leucine incorporated into cells exposed to LipofectAMINE 2000-dsRNA complex/[<sup>3</sup>H]-leucine incorporated into cells exposed to LipofectAMINE 2000 alone×100.</p
Effect of extracellular potassium on IL-1 processing and release.
<p>A) Bone marrow-derived macrophages were plated in triplicate wells in 12-well plates and primed with 50 ng/ml of LPS for 4 h. Cells were then incubated in medium containing 130 mM NaCl/5 mM KCl or 5 mM KCl/130 mM NaCl in the presence of absence of 0.01 µg/mL ricin, 25 µg/mL cycloheximide, 10 µg/mL, 10 µg/mL emetine, 75 µg/mL puromycin, 0.2 µg/mL pactamycin, 10 µg/mL anisomycin, 3.4 µM nigericin, or 5 mM ATP for 4 h. Medium was collected, and p17 IL-1 was determined by ELISA. B) Macrophages were plated, primed with LPS and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 5 mM ATP or 3.4 µM nigericin for 4 h. Proteins were precipitated from the media with TCA and analyzed by Western blotting. C) Macrophages were plated, primed with LPS, and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 10 ng/mL ricin, 10 µg/mL emetine, or 25 µg/mL cycloheximide for 4 h. Proteins were precipitated with TCA and analyzed by Western blotting.</p
Proteasome inhibitors block processing and release of IL-1ß.
<p>A) LPS-primed WT BMDM were incubated in control medium or medium containing 10 ng/ml ricin or 25 µg/ml cycloheximide for 4 h. MG-132 (30 µM) or Bortezimib (0.5 µM) was included as indicated. Secreted IL-1ß was measured by ELISA in triplicate wells. B) LPS-primed WT BMDM were incubated in the presence or absence of MG-132 for 4 hours, in the presence or absence of inhibitors of protein synthesis, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting. C) LPS-primed or unprimed WT BMDM were or exposed to MSU, MG-132, or both for 4 h, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting.</p
Epidemiological model including two modes of immunisation. Model setup and outcomes.
<p>(A) Illustration of the SIRM (Susceptible-Infectious-Removed-iMmune) model, with (B) corresponding state changes and transition rates under which ants change their states. The dotted line in (A) illustrates the influence of infectious individuals (<i>I</i>) on the state change rate from susceptible (<i>S</i>) to initially immunised (<i>M<sub>i</sub></i>) ants for passive immunisation. (C,D) Model predictions for the proportions of individuals in the different states over time, comparing passive (C) and active (D) immunisation. Passive immunisation allows for a higher number of immune individuals (<i>M<sub>i</sub></i> and entering the <i>M<sub>l</sub></i> state, pale and dark blue dashed lines), whereas active immunisation leads to a faster elimination of the disease (infectious [<i>I</i>, black solid line] individuals go to 0) and a lower death rate in the colony (<i>R</i>, red solid line), despite the fact that disease spread from the first exposed ants can only occur in the active immunisation scenario. Immunisation is transient so that <i>M<sub>l</sub></i> individuals become susceptible (<i>S</i>, green dotted line) over time for both passive and active immunisation.</p