Knockdown of P-gp measured at mRNA level by qRT-PCR.

<p>The cells were transfected with only chitosan (mock, M) or nanoparticles having N/P 30 and P-gp targeting (T) or non-targeting (NT) siRNA concentrations of 100 nM. Cells were also treated with naked siRNA (siRNA). Data represents mean values ± s.d., n = 3.</p

The effect of P-gp knockdown on R123 efflux.

<p>Intracellular levels of R123 as a function of A) siRNA concentration and B) days post-transfection. The relative levels of R123 are expressed as the median FI of the cells. The cells were transfected with nanoparticles having N/P 30 and P-gp targeting (T) or non-targeting (NT) siRNA concentrations of 100 nM. Data represents mean values ± s.d., n = 3. C) Representative histograms of R123 fluorescence from flow cytometry analysis of untreated cells or cells transfected with nanoparticles having N/P 30 and a siRNA concentration of 100 nM at one to five days post-transfection. D) Representative CLSM images after incubation with R123 post-transfection with a) T or b) NT siRNA or c) untreated cells. The cells were transfected with nanoparticles having N/P ratios of 30 and a siRNA concentration of 100 nM. The cellular plasma membranes were stained with CellMask Deep Red (blue) and R123 fluorescence is indicated with the green color. The bar size is 20 µm.</p

Particle concentrations measured by nanoparticle tracking analysis.

<p>The samples consisted of complexes in formulations having N/P 10, 30 or 60 and a siRNA concentration of 500 nM. Data represents mean values ± s.d., n = 3.</p

The qRT-PCR primers used in the study.

<p>The qRT-PCR primers used in the study.</p

The effect of P-gp knockdown on doxorubicin efficacy and delivery.

<p>A) Metabolic activity of cells after two days of incubation with doxorubicin. The cells were transfected with nanoparticles having N/P 30 and P-gp targeting (T) or non-targeting (NT) siRNA concentrations of 50 or 100 nM. Data represents mean values ± s.d., n = 4. B) Intracellular uptake and accumulation of doxorubicin expressed as the median FI of the cells. The cells were transfected with nanoparticles having N/P 30 and T or NT siRNA concentrations of 100 nM. Data represents mean values ± s.d., n = 3. C) Representative histograms of doxorubicin fluorescence from flow cytometry analysis of untreated or transfected cells. D) Representative CLSM images after transfection with a) T or b) NT siRNA or c) untreated cells. E) Enlarged images of RBE4 nuclei. The cells were transfected with nanoparticles having N/P 30 and a siRNA concentration of 100 nM. Doxorubicin fluorescence is indicated with the green color. The bar size is 20 µm.</p

Molecular characterization of the chitosan used in the study.

<p>The weight and number average of the molecular weight (M_w, M_n) and the polydispersity index (PDI) were analyzed by SEC-MALLS. The fraction of <i>N</i>-acetylated units (F_A) was determined by ¹H NMR.</p

Chitosan-mediated siRNA uptake in RBE4 cells.

<p>A) Levels of internalized Alexa-647 conjugated siRNA at different nanoparticle N/P ratios and siRNA concentrations expressed as the median fluorescence intensities (FI) of the analyzed cells. Data represents mean values ± s.d., n = 3. B) Representative histograms of siRNA fluorescence from flow cytometry analysis of untreated cells, cells with added naked siRNA or transfected with nanoparticles having N/P 10, 30 or 60 and a siRNA concentration of 100 nM. C) Representative CLSM images of a) untreated cells, b) cells with added naked siRNA or nanoparticles having N/P c) 10, d) 30 or e) 60 and a siRNA concentration of 100 nM. The cellular plasma membrane was stained with CellMask Orange (blue) and the fluorescent siRNA is indicated with the red color. The bar size is 20 µm.</p

Cytokine Profiles in Human Metapneumovirus Infected Children: Identification of Genes Involved in the Antiviral Response and Pathogenesis

<div><p>Human metapneumovirus (hMPV) causes severe airway infection in children that may be caused by an unfavorable immune response. The nature of the innate immune response to hMPV in naturally occurring infections in children is largely undescribed, and it is unknown if inflammasome activation is implicated in disease pathogenesis. We examined nasopharynx aspirates and blood samples from hMPV-infected children without detectable co-infections. The expression of inflammatory and antiviral genes were measured in nasal airway secretions by relative mRNA quantification while blood plasma proteins were determined by a multiplex immunoassay. Several genes were significantly up-regulated at mRNA and protein level in the hMPV infected children. Most apparent was the expression of the chemokine IP-10, the pro-inflammatory cytokine IL-18 in addition to the interferon inducible gene ISG54. Interestingly, children experiencing more severe disease, as indicated by a severity index, had significantly more often up-regulation of the inflammasome-associated genes IL-1β and NLRP3. Overall, our data point to cytokines, particularly inflammasome-associated, that might be important in hMPV mediated lung disease and the antiviral response in children with severe infection. Our study is the first to demonstrate that inflammasome components are associated with increased illness severity in hMPV-infected children.</p></div

Expression of pro-inflammatory genes in nasopharyngeal aspirates from controls or children infected with hMPV genotype A2 or B2.

<p>Relative levels of A) IP-10, B) TNF-α, C) IL-6 and D) ISG54 mRNA expression. Data was plotted as box-plots where the box represents 50% of the values, the horizontal line indicates the median, whiskers show the maximum and minimum values, and the closed circles indicate outliers. Asterisk indicates statistical significant difference (P<0.05, ANOVA test).</p

Individual variation in the expression of inflammasome-associated genes in hMPV infected children.

<p>Relative levels of A) IκBα, B) IL-1β and C) NLRP3 mRNA expression relative to the controls. The median level of expression for the controls is indicated with the dotted line. Asterisk indicates statistical significant difference (P<0.05, ANOVA test).</p