Binding of serum IgG to intact pneumococci.

<p>Sera from mice immunized with two doses of the indicated formulations were tested for the ability to bind to pneumococcal strains expressing PspA from clades 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E). Results are shown as fluorescence intensity histograms and are representative of two experiments using sera from independent immunizations.</p

Immunophenotyping of cells recovered in BALF collected 12 hours after lethal challenge with ATCC6303.

<p>Mice were immunized with 2 doses of the indicated formulations and challenged with pneumococcal strain ATCC6303. BALF was collected 12 hours after challenge and recovered cells were immunophenotyped by flow cytometry. Percentages of alveolar macrophages (AM—F4/80+ CD11c+ CD11b-) (A), exudate macrophages (EM—F4/80+ CD11c- CD11b+) (B), B cells (F4/80- B220+) (C), CD4+ T cells (F4/80- CD4+) (D), CD8+ T cells (F4/80- CD8+) (E) and neutrophils (F4/80- Ly6G+) (F) are shown. Means and standard errors of each group are shown.</p

Cytokine/chemokine levels in BALF collected 12 hours after lethal challenge with ATCC6303.

<p>Mice were immunized with 2 doses of the indicated formulations and challenged with pneumococcal strain ATCC6303. Levels of IL-6 (A), TNF-α (B), KC/CXCL1 (C) and MIP-2/CXCL2 (D) were determined in BALF collected 12 hours after challenge by Luminex. Naive mice were not immunized nor challenged. * indicates statistically significant difference (One-way ANOVA, Tukey’s Multicomparison Test). Symbols represent each individual. Means±standard errors are shown. Representative of two independent experiments.</p

Induction of anti-PspA4Pro antibodies in BALF by mucosal immunization targeting the lungs.

<p>The induction of anti-PspA4Pro IgG (A) and IgA (B) antibodies in BALF from mice inoculated with the indicated formulations was determined by ELISA. Mice were inoculated with 2 doses of the formulations. Control mice were inoculated sc with saline or PspA4Pro. <i>A</i>_<i>405</i> nm of samples diluted 1:2 is shown. * indicates statistically significant difference with saline (One-way ANOVA, Tukey’s Multicomparison Test). Symbols represent each individual. Means±standard errors are shown.</p

Pneumococcal load in BALF collected 24 hours after non-lethal challenge with EF3030.

<p>Mice were immunized with 2 doses of the indicated formulations and challenged with pneumococcal strain EF3030. Pneumococcal load in BALF was determined 24 hours after challenge. Symbols represent each individual. Means±standard errors are shown.</p

Induction of serum anti-PspA4Pro IgG antibodies by mucosal immunization targeting the lungs.

<p>The induction of anti-PspA4Pro IgG antibodies in sera from mice inoculated with the indicated formulations was determined by ELISA. Mice were inoculated with 1 (A) or 2 (B and C) doses of the formulations. Control mice were inoculated sc with saline or PspA4Pro. Log₁₀ anti-PspA4Pro IgG titers (A and B) and log₁₀ anti-PspA4Pro IgG1 titer/log₁₀ anti-PspA4Pro IgG2a titer ratios (C) are shown. * indicates statistically significant difference with saline and # indicates statistically significant difference with PspA4Pro sc (One-way ANOVA, Tukey’s Multicomparison Test for A and B; Unpaired t-test for C). Symbols represent each individual. Means±standard errors are shown. Representative of at least two independent experiments.</p

Inflammatory responses are impaired in monocytes from CF patients.

<p>Monocytes from healthy volunteers (control, gray bars, n = 10) and from CF patients (CF, solid bars, n = 20) were cultured in the presence of 10 ng/ml LPS for indicated times. Cells were harvested, total RNA isolated and cDNA synthesized. TNFα (A) and IL-6 (B) mRNA levels were analyzed by real time Q-PCR. The ratios [TNFα]/[18S] and [IL-6]/[18S] are depicted. **, p<0.01 with respect to healthy controls (C) Concentrations of TNFα were determined in supernatants of cultures of human monocytes from healthy volunteers (control, gray bars, n = 10) and CF patients (CF, solid bars, n = 20), stimulated or not with 10 ng/ml LPS. *, p<0.05 with respect to healthy controls. (D) Monocytes isolated from healthy volunteers (control, gray bars, n = 10) and CF patients (CF, solid bars, n = 20) were stained with anti-CD14-PE, anti-CD16b or anti-CD1a antibodies and then analyzed by flow cytometry; the fraction of cells stained with each antibody is given.</p

CF but not COPD monocytes fail to induce TREM-1 expression after LPS stimulation.

<p>(A) Cultures of human monocytes from healthy volunteers (control, gray bars, n = 10) CF patients (CF, solid bars, n = 20) and COPD patients (COPD, hatched bars, n = 10) were treated with 10 ng/ml LPS for indicated times. Next, cells were harvested and stained with anti-TREM-1-FITC and analyzed by flow cytometry. The fraction of positive cells is given as % of total cells (A) and MFIs (B). The percentage of CD14 and TREM-1 positive cells was analyzed in monocytes from five of each of the studied groups (i.e., CF patients, COPD patients and healthy individuals; all samples were randomly selected), treated or not <i>ex vivo</i> with LPS for 1 hour. The flow cytometry analysis of these cells simultaneously stained with CD14-APC and TREM-1-FITC is shown (C). Supernatants from the same experiment (shown in panels A and B) were centrifuged at 400×g to remove detached cells and possible cellular debris. The concentration of sTREM-1 in the supernatants was analyzed with a commercial ELISA. The mean concentration of sTREM-1 in ng/ml is given (D). *, p<0.05 and **, p<0.01 with respect to similarly treated monocytes from healthy individuals.</p

Expression levels of TREM-1 and sTREM-1 are low in CF monocytes.

<p>(A) Monocytes isolated from healthy volunteers (controls, gray bars, n = 10) and CF patients (CF, solid bars, n = 20) were stained with anti–TREM-1-FITC and then analyzed by flow cytometry; the fraction of cells stained is given as % of total cells (left scale) and as <u>m</u>ean <u>f</u>luorescence <u>i</u>ntensity (MFI, right scale). *, p<0.05 with respect to healthy controls. (B) A typical histogram obtained from flow cytometric analysis of TREM-1 expression is shown (I, isotype; CF, CF patients; Control, healthy volunteers). (C) The concentration of sTREM-1 in the supernatants of the same cultures was analyzed with a commercial ELISA. The mean concentration of sTREM-1 is represented. (D) Supernatants from monocyte cultures (one from a healthy volunteer (control) and two from randomly selected CF patients) were centrifuged at 400×g to remove detached cells and possible cellular debris. A Western blot analysis of these supernatants was performed using an anti–TREM-1 commercial antibody that recognizes the extra-cellular domain of TREM-1. Human monocytes challenged with LPS for six hours were used as positive control (+). Notice detection of a band of approximately 27 kDa in this condition (marked with an arrow). Loading controls are also shown (membrane stained with Ponceau red, lower panel). The results of a triplicated experiment are shown.</p

Elements of the LPS receptor complex are expressed at similar levels in control and CF monocytes.

<p>Monocytes isolated from healthy volunteers (control, gray bars, n = 10) and CF patients (CF, solid bars, n = 20) were stained with anti-CD14 and anti-TLR4/MD2 and then analyzed by flow cytometry; the fraction of cells stained is given (A and B respectively). Typical histograms are shown in the insets (I, isotype; Control, healthy volunteers; CF, Cystic Fibrosis).</p