Co-treatment of A549 cell with IL-1β and <i>Viscum album</i> inhibits the cytokine-induced COX-2 expression.

<p>A549 cells were treated with IL-1β (10 ng/ml) and two different concentrations of <i>Viscum album</i> Q Spez preparation for 18 hours. Cytosolic COX-2 was measured using flow cytometric analysis. <i>Viscum album</i> is added to the cells either from the beginning of the experiment along with IL-1β (co-treatment) or after 14 hours of IL-1β induction (post-treatment). Percentage COX-2 expression as measured in intracellular staining by flow cytometry (A) and mean fluorescence intensity (MFI) of COX-2 expression (B) is shown. Results are mean ±SEM of 4 independent experiments (**<i>p</i><0.01; ***<i>p</i><0.001).</p

Inhibition of PGE2 secretion by <i>Viscum album</i> in A549 cells.

<p>Cells were treated with IL-1β (10 ng/ml) and increasing concentrations of VA Qu Spez for 18 hours. PGE2 was analyzed in culture supernatants by EIA. Results are mean±SEM from 4 independent experiments (*<i>p</i><0.05 versus control cells, **<i>p</i><0.05 versus cells treated with IL-1β, analyzed by paired Student-t-test).</p

Effect of <i>Viscum album</i> on COX-1 expression in A549 cells.

<p><b>A and B.</b> A549 cells were treated with increasing concentrations of VA Qu Spez in presence of IL-1β (10 ng/ml) for 18 hours. Protein expression of COX-1 was analyzed by western blot. A representative western-blot depicting the effect of VA on COX-1 expression under IL-1β-stimulated culture conditions (A). Relative expression (mean ± SEM) of COX-1 protein from three independent experiments as quantified by densitometry (ratio of density of COX-1: that of β-actin). ns, non-significant (B). <b>C.</b> Cells were cultured either in medium alone or treated with increasing concentrations of VA Qu Spez without IL-1β for 18 hours. 20 µg of total cellular protein was separated by 10% SDS-PAGE followed by western blotting to analyze the expression of COX-1. Results are representative of two experiments.</p

Effect of <i>Viscum album</i> on the stability of COX-2 protein as determined by western blot.

<p>Confluent A549 cells were treated with IL-1β in the presence and absence of VA Qu Spez in dose dependent concentrations in μg/ml. Cells were harvested at different time intervals after blocking the protein synthesis with cyclohexamide (10 μg/ml) for 90 minutes till 11 hours. COX-2 expression was measured by western blot using the cytosolic extracts. (A), inhibition of COX2 protein synthesis by VA at 18 hours. (B) (C) (D) are the representative western blots after 90 minutes, 5 hours and 11 hours respectively showing level of COX-2 expression after cyclohexamide treatment with or without <i>Viscum album</i>. β-actin was used as an internal control. All blots are representative of three independent experiments and the densitometry values for each band are mentioned below the representative blots.</p

Effect of <i>Viscum album</i> on the stability of COX-2 protein as analyzed by flow cytometry.

<p>A549 cells were stimulated with IL-1β for 90 minutes with or without VA Qu Spez. Cells were harvested at different time intervals after blocking the protein synthesis with cyclohexamide (10 μg/ml) for 90 minutes till 11 hours. Normalised percentage COX-2 expression as measured in intracellular staining by flow cytometry (A) and mean fluorescence intensity (MFI) of COX-2 expression (B) is shown. Data is representative of mean ±SEM of three independent experiments.</p

Inhibition of cytokine-induced COX-2 protein expression by <i>Viscum album</i> in A549 cells.

<p>A549 cells were either cultured in medium alone or treated with IL-1β (10 ng/ml) with or without increasing concentration of VA Qu Spez for 18 hours. Using 20 µg of total cellular protein, expression of COX-2 was analyzed by western blotting. <b>A.</b> A representative western-blot depicting the effect of VA on expression of IL-1β-induced COX-2. <b>B.</b> Relative expression (mean ± SEM) of COX-2 protein from four independent experiments as quantified by densitometry (ratio of density of COX-2: that of β-actin). *<i>P</i><0.05 versus control cells and ** <i>p</i><0.05 versus IL-1β-stimulated cells as analyzed by paired Student-t-test. <b>C.</b> Kinetics of COX-2 protein expression upon treatment of A549 cells with various doses of VA. The cells were either cultured in medium alone or stimulated with IL-1β with our without VA for 12, 18, 24 and 36 hours. Expression of COX-2 protein was analyzed by western blotting and relative expression of COX-2 protein was quantified by densitometry (ratio of density of COX-2: that of β-actin). Results are representative of two experiments.</p

Effect of VA Q Spez on IL-1β-induced COX-2 transcription.

<p>Total cellular RNA was isolated from A549 cells that were cultured either in medium alone or stimulated with IL-1β (10 ng/ml) with or without various concentrations of VA for 18 hours. mRNA expression of COX-2 was analyzed by semi quantitative RT-PCR using COX-2-specific primers. Amplification of a house keeping gene GAPDH is used as an internal control. (A) PCR products were separated on 1% agarose gel and a representative gel picture is shown. (B) Relative expression (mean ± SEM) of COX-2 mRNA from six independent experiments as quantified by densitometry (ratio of density of COX-2: that of GAPDH). * P<0.05 analyzed by paired Student-t-test, ns refers to non-significant, compared to IL-1β stimulated cells.</p

Oligonucleotide primers used in the study.

<p>Oligonucleotide primers used in the study.</p

Selective inhibition of COX-2 expression by <i>Viscum album</i>.

<p>Quantitative comparison between expression of COX-1 and COX-2 proteins as revealed by western blot. Protein expression was quantified by densitometry and expressed as percentage protein expression normalized to that in cells stimulated with IL-1β. Results (mean ± SEM) obtained from 3 independent experiments are presented. * <i>P</i><0.05 versus control cells for COX-2 and ** <i>p</i><0.05 versus IL-1β-stimulated cells for COX-2 as analyzed by paired Student-t-test. The values for COX-1 are non-significant.</p

Treatment with IVIg is associated with a transient decrease in levels of PFR-MCA hydrolyzing IgG.

<p>IgG was purified from the plasma of patients who received IVIg therapy prior to transplantation (full circles) and from patients who did not received IVIg (empty circles). Plasma had been collected prior to renal transplant (D0) and 3 (M3), 12 (M12) and 24 (M24) months after renal transplant. IgG (66.67 nM) was incubated with PFR-MCA (100 µM), a peptide chromogenic substrate, for 24 hr at 37°C. The amount of hydrolysis was quantified by measuring the fluorescence of the leaving MCA moiety, and is expressed in femtomoles of substrate hydrolyzed per minute per picomoles of IgG. Pooled normal human IgG was used as a control source of IgG. Panel A depicts the raw results as scatter dot plots. Panel B depicts the evolution of the mean ± SEM levels of PFR-MCA-hydrolyzing IgG in the two groups of patients with time (*: P = 0.004). The dotted line represents the hydrolysis of PFR-MCA by normal pooled human IgG (mean of 29 measurements; Coefficient of variation: 0.29). Panel C depicts the levels of PFR-MCA-hydrolyzing IgG in patients treated with anti-thymocyte globulins (ATG, full squares) or not (empty squares), as measured in plasma collected 3 months post-transplantation.</p