Study protocol.

<p>OVA: ovalbumin; CNP: Degussa-FW2 (13 nm) carbon nanoparticles; MCH: methacholine; FOT: forced oscillation respiratory mechanics measurements; EELV: end-expiratory lung volume measurements. Brown-Norway rats underwent 4 treatments; OVA-sensitization, CNP tracheal instillation, Both OVA-sensitization and CNP treatment, and sham sensitization and tracheal instillation of vehicle. Equal treatment groups were exposed to either air or NO₂.</p

Maximal airway and tissue responses to OVA provocation, relative to the recovery level from MCH. Data are means ± SE (n = 6 per group); *: p<0.05 vs. Air+Saline within condition; #: p<0.05 vs. Air+OVA; £: vs. Air+OVA+CNP, by ANOVA.

<p>The responses in Raw were significantly larger in the NO₂-exposed and sensitized animals. In the group concomitantly treated with CNP during sensitization (NO2+OVA+CNP), the maximal Raw change in response to OVA provocation was smaller compared to NO2+OVA.</p

BALF Protein Levels of TH1 and TH2 Cytokines.

<p>Values are means ± SE (n = 6 per group);</p>a<p>p<0.05 vs. Saline within condition;</p>b<p>p<0.05 vs. CNP within condition;</p>c<p>p<0.05 vs. OVA within condition;</p>d<p>p<0.05 vs. Air within treatment, by ANOVA. NO₂ exposure significantly increased both KC/GRO and IL-5 compared to air-exposed animals. Individual increases in KC/GRO were significantly higher vs. Air-Saline, in the Air-CNP, NO2+Saline and NO2+OVA groups.</p

BALF Cytology.

<p>Values are means ± SE (n = 6 per group);</p>a<p>p<0.05 vs. Saline within condition;</p>b<p>p<0.05 vs. CNP within condition;</p>c<p>p<0.05 vs. OVA within condition;</p>d<p>p<0.05 vs. Air within treatment, by two way ANOVA; <b>AM</b>: alveolar macrophages. Exposure to NO₂ alone did not change the profile of BAL cytology. OVA-sensitization significantly increased BAL eosinophil counts. CNP exposure by itself increased the number of neutrophils, without a significant difference between Air and NO₂-exposed animals. Exposure to NO₂ in OVA-sensitized animals significantly increased lymphocyte counts and tended to elevate BALF eosinophilia.</p

Representative histological images of HES (left columns), PAS (middle columns) and Masson’s trichrome stain (right columns).

<p>Original magnification: ×100. Exposure to NO₂, resulted in inflammatory changes, including infiltration in the bronchial submucosa, perivascular areas and the surrounding alveolar septa (left columns: arrow). Mild to moderate goblet cell hyperplasia (middle columns: arrow), and the presence of mucus secretions inside the bronchial lumen (middle columns: star), following NO₂ exposure in the OVA-sensitized groups. Increased collagen deposition in the bronchial wall associated with exposure to NO₂ was found on Masson’s trichrome (middle columns: arrow).</p

IL-17A levels in BAL fluid.

<p>Data are means ± SE; *: p<0.05 vs. Saline control within condition; #: p<0.05 vs. CNP within condition; £: vs. OVA within condition, &: vs. OVA within treatment, by ANOVA.</p

Changes in end-expiratory lung volume (EELV).

<p>Data are means ± SE (n = 6 per group); *: p<0.05 vs. 7 week old Control; #: p<0.05 vs. 11-week old Air+Saline by ANOVA. The EELV was significantly lower in the NO2+Saline vs. Air+Saline animals. Also, EELV was significantly reduced by sensitization to allergen, CNP treatment or both, independent of exposure to NO₂. Comparison of EELV at 11 weeks vs. non-exposed controls at 7 weeks (n = 32) revealed that EELV significantly increased over the 4-week exposure period in all groups with the exception of NO2+OVA and NO2+OVA+CNP.</p

Serum Protein Levels of TH1 and TH2 Cytokines.

<p>Values are means ± SE (n = 6);</p>a<p>p<0.05 vs. Saline within condition;</p><p>bp<0.05 vs. CNP within condition;</p>c<p>p<0.05 vs. OVA within condition;</p>d<p>p<0.05 vs. Air within treatment, by ANOVA;</p>$<p>Below detection limit. In serum, the TH2 cytokines IL-4, IL-5 and IL-13, were significantly increased in the NO2+OVA group, but not in the NO2+OVA+CNP group. Exposure to NO₂ significantly increased serum TNF-α expression, in all of the exposed groups, which was remarkably higher in both of the OVA-sensitized groups; NO2+OVA and NO2+OVA+CNP vs. NO2+Saline and NO2+CNP. In the NO2+OVA and NO2+OVA+CNP groups, the levels of IL1-b were significantly increased compared to their air-exposed counterparts. The combination of NO₂ exposure and CNP treatment significantly increased the IFN-g levels; however, the level of this cytokine was again significantly lower in the NO 2+OVA+CNP group. The KC/GRO levels were increased following NO₂ exposure, with substantial elevations in NO2+OVA and NO2+OVA+CNP groups compared to Air+Saline, with notable within-group variability.</p

Total serum IgE levels.

<p>Data are means ± SE; *: p<0.05 vs. Saline control within condition; #: p<0.05 vs. CNP within condition; £: vs. OVA+CNP within condition, by ANOVA.</p

Size distribution of TiO₂ suspension in NaCl determined by dynamic light scattering.

<p>Size distribution of TiO₂ suspension in NaCl determined by dynamic light scattering.</p