Adjuvant effect of DMXAA can improve potency of influenza SV vaccine and protect mice from a lethal challenge.

<p>C57BL/6 mice were immunized twice i.d. with 0.75 µg SV and 100 µg DMXAA, and their sera were assessed for anti-SV (A) tIgG, (B) IgG1 and (C) IgG2c antibodies. ** <i>P</i><0.01 by Students t-test. Results presented are pooled from two separate experiments. (D) C57BL/6 mice that had received two i.d. injections of PBS (open square), 100 µg DMXAA alone (open circle), 0.75 µg SV alone (grey-filled circle) or SV plus DMXAA (black filled circle) were challenged with a lethal dose of A/Puerto Rico/8/34 (PR) (H1N1) 7 days after the final immunization (n = 6 mice per experimental group). Results presented are pooled from two separate experiments. Survival rates were recorded daily and statistical analyses were performed using the log-rank (Mantel-Cox) test where ** and *** denotes p<0.01 and p<0.001 respectively (E) The rate of weight-loss by the challenged mice were monitored and presented as an average percentage of the initial base weight ± standard error. * denotes p<0.05 vs SV alone by Student's T-test.</p

Adjuvant effects of DMXAA require type-I-IFN responses induced by IRF3 activation.

<p>Anti-OVA (A) tIgG antibody responses of WT C57BL/6, <i>Ifnar^-/-</i> and <i>Irf3^-/-</i> mice immunized twice i.d. with 10 µg OVA plus 100 µg DMXAA. (B) tIgG, (C) IgG1 and (D) IgG2a antibody titers against OVA in WT BALB/c and <i>Il-33^-/-</i> mice immunized twice i.d. with 10 µg OVA plus 100 µg DMXAA. Results presented are pooled titers from two separate experiments. <i>In-vitro</i> cultured DCs derived from WT, <i>Ifnar^-/-</i> and <i>Irf3^-/-</i> mice were stimulated with DMXAA (2.5 µg/ml), LPS (1 µg/ml) or lipofectamine complexed c-di-GMP (10 µg/ml) for 6 h before the supernatant were collected and analysed for (E) IFNβ, (F) IL-6 and (G) TNFα secretion and CD11c⁺ cells were analysed for CD86 expression (H). Results presented are average of triplicate conditions ± SD and are representative of three separate experiments. *** <i>P</i><0.001 one-way ANOVA with Bonferroni's post-test.</p

DMXAA acts as a potent adjuvant.

<p>(A) Anti-OVA tIgG titers of C57BL/6 mice immunized with 100 µg OVA plus the indicated doses of DMXAA (µg). (B) Anti-OVA tIgG titers of C57BL/6 mice 21 days and 150 days after immunization with 100 µg OVA and 100 µg DMXAA. (C–E) C57BL/6 mice were immunized twice i.d. with 100 µg OVA plus DMXAA (100 µg), Alum (665 µg) or CpG DNA (25 µg) and the induction of (C) tIgG, (D) IgG1 and (E) IgG2c antibody responses against OVA were assessed. (F) IFN-γ secretion from splenocytes of immunized mice that were stimulated for 48 h with CD4 and CD8 OVA peptides and whole OVA protein. (G) Anti-OVA tIgG titers of C57BL/6 mice injected i.v. with 200 µg anti-CD4 (GK1.5) antibodies prior to immunization with 100 µg OVA and 100 µg DMXAA. Results presented are representatives of three separate experiments. *<0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 by Students t-test when comparing between two groups and one-way ANOVA with Bonferroni's post-test when comparing three or more groups.</p

Comparison of pulmonary inflammatory responses following intratracheal instillation and inhalation of nanoparticles

<p>In order to examine whether intratracheal instillation studies can be useful for determining the harmful effect of nanoparticles, we performed inhalation and intratracheal instillation studies using samples of the same nanoparticles. Nickel oxide nanoparticles (NiO) and titanium dioxide nanoparticles (TiO₂) were used as chemicals with high and low toxicities, respectively. In the intratracheal instillation study, rats were exposed to 0.2 or 1 mg of NiO or TiO₂. Cell analysis and chemokines in bronchoalveolar lavage fluid (BALF) were analyzed from 3 days to 6 months following the single intratracheal instillation. In the inhalation study, rats were exposed to inhaled NiO or TiO₂ (1.65, 1.84 mg/m³, respectively) for 4 weeks. The same endpoints were examined from 3 days to 3 months after the end of exposure. Inhalation of NiO induced an increase in the number of neutrophils in BALF and concentrations of cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2 and heme oxygenase (HO)-1. Intratracheal instillation of NiO induced persistent inflammation and upregulation of these cytokines was observed in the rats. However, inhalation of TiO₂ did not induce pulmonary inflammation, and intratracheal instillation of TiO₂ transiently induced an increase in the number of neutrophils in BALF and the concentrations of CINC-1, CINC-2 and HO-1. Taken together, a difference in pulmonary inflammation was observed between the high and low toxicity nanomaterials in the intratracheal instillation studies, as in the inhalation studies, suggesting that intratracheal instillation studies may be useful for ranking the harmful effects of nanoparticles.</p