Effects of BZ on hypoxia-selected K562 cells.

<p>Cells from day-7 hypoxic LC1 treated with BZ at day 6 as indicated were transferred into normoxic LC2 (3×10⁴ viable cells/ml) and trypan blue-negative cells counted at the indicated times of incubation in LC2. Values represent means±S.E.M. of data from 3 independent experiments.</p

Induction of apoptosis by BZ in hypoxia.

<p>(<b>A</b>) Total cell lysates in Laemmli buffer were subjected to immuno-blotting with the indicated antibodies. Anti-H4 was used to verify equalization of protein loading. One representative experiment out of 3 is shown. (<b>B</b>) Percentages cells undergone “early” (annexin-V+/PI-) or “late” (annexin-V+/PI+) apoptosis, as determined by flow-cytometry. Values are means±S.E.M. of 3 independent experiments.</p

Effects of BZ on hypoxia-resistant K562 cell subsets.

<p>Cells treated as indicated in hypoxic or normoxic LC1 (established at 3×10⁴ viable cells/ml) were transferred at day 2 (<b>A</b>) or day 7 (<b>B</b>) into normoxic LC2 (3×10⁴ viable cells/ml) and trypan blue-negative cells counted at the indicated times of incubation in LC2. Values represent means±S.E.M. of data from 3 independent experiments.</p

Effects of BZ on BCR/Abl protein expression in hypoxia.

<p>Total cell lysates in Laemmli buffer were subjected to immuno-blotting with an anti-Abl antibody. Anti-vinculin and anti-ERK1/2 antibodies were used to verify equalization of protein loading. One representative experiment out of 3 is shown.</p

Effects of Bortezomib (BZ) on K562 cell bulk.

<p>Cultures were treated or not with a single dose of 0.5 nM BZ from time 0 to day 7 or from day 1 to day 7 and trypan blue-negative cells counted at the indicated times. Values represent means±S.E.M. of data from 3 independent experiments. (<b>A</b>): hypoxia (∼0.1% O₂); (<b>B</b>): normoxia (21% O₂).</p

Effect of capsular polysaccharides from ST258 KP strains on caspase-1 activation.

<p>Monocytes were cultured at 2x10⁶/ml with purified 5 μg/ml of capsular polysaccharides from ST258-KP strains for 7 hours. Cells were lysed and immunoblotted with antibodies specific caspase-1 (p20) and tubulin. Results from one representative experiment out of three performed are shown. Histograms show the results of densitometric analysis (mean ± SE) from three different experiments.</p

IL-1β production by monocytes and MDC cultured with strains of <i>K</i>.<i>pneumoniae</i>-KPC with different capsular phenotype.

<p>IL-1β production by monocytes and MDC cultured with strains of <i>K</i>.<i>pneumoniae</i>-KPC with different capsular phenotype.</p

Effect of <i>K</i>. <i>pneumoniae</i> strains on the function of dendritic cells.

<p>MDC or monocytes were cultured for 16 hours with UV-treated bacteria or with LPS (400 ng/ml) as a control. At the end of incubation, MDC were stained with anti-CD80-FITC, anti-CD86-APC, anti-HLA-DR-PE or isotype control and analyzed by cytofluorimetric analysis. Results are expressed as mean fluorescence intensity. Data from 6 experiments (mean ± SE) are shown. Statistical analysis was performed by One-Way ANOVA and <i>p</i> ≤ 0.05 was considered significant; no significant differences were revealed.</p

Th1 differentiation of CD4⁺ T lymphocytes induced by <i>K</i>. <i>pneumoniae</i> strains.

<p>PBMC from 6 healthy donors were cultured at 10⁶/ml for 7 days with UV-inactivated bacterial cells at 1:10 ratio. Cells were recovered at the indicated times, stained with anti-CD4-APC and anti-IFNγ-PerCP and analyzed by ACCURI instrument using the CflowPlus software to process the data. The area of positivity was determined by using an isotype-matched control mAb. Representative scatter plots at day 5 are shown. Data are expressed as percentage of CD4⁺IFNγ⁺ (mean ± SE).</p

Differences in Inflammatory Response Induced by Two Representatives of Clades of the Pandemic ST258 <i>Klebsiella pneumoniae</i> Clonal Lineage Producing KPC-Type Carbapenemases

<div><p>ST258-<i>K</i>. <i>pneumoniae</i> (ST258-KP) strains, the most widespread multidrug-resistant hospital-acquired pathogens, belong to at least two clades differing in a 215 Kb genomic region that includes the cluster of capsule genes. To investigate the effects of the different capsular phenotype on host-pathogen interactions, we studied representatives of ST258-KP clades, KKBO-1 and KK207-1, for their ability to activate monocytes and myeloid dendritic cells from human immune competent hosts. The two ST258-KP strains strongly induced the production of inflammatory cytokines. Significant differences between the strains were found in their ability to induce the production of IL-1β: KK207-1/clade I was much less effective than KKBO-1/clade II in inducing IL-1β production by monocytes and dendritic cells. The activation of NLRP3 inflammasome pathway by live cells and/or purified capsular polysaccharides was studied in monocytes and dendritic cells. We found that glibenclamide, a NLRP3 inhibitor, inhibits more than 90% of the production of mature IL-1β induced by KKBO1 and KK207-1. KK207-1 was always less efficient compared to KKBO-1 in: a) inducing NLRP3 and pro-IL-1β gene and protein expression; b) in inducing caspase-1 activation and pro-IL-1β cleavage. Capsular composition may play a role in the differential inflammatory response induced by the ST258-KP strains since capsular polysaccharides purified from bacterial cells affect NLRP3 and pro-IL-1β gene expression through p38MAPK- and NF-κB-mediated pathways. In each of these functions, capsular polysaccharides from KK207-1 were significantly less efficient compared to those purified from KKBO-1. On the whole, our data suggest that the change in capsular phenotype may help bacterial cells of clade I to partially escape innate immune recognition and IL-1β-mediated inflammation.</p></div