Hypothesis and Specific Aims: Our hypothesis is that the inhalation of ultrafine carbon particles exacerbates allergic asthma in susceptible individuals. The four specific Aims being used to test this hypothesis are:

1. Induce immune responses in the lungs of dogs having high and low susceptibility for developing allergic diseases.
2. Determine the level of ragweed allergen exposure that produces a 20-50% increase in airway resistance in the susceptible dogs.
3. Determine if the inhalation of ultrafine carbon particles alone induces airway responses in dogs having allergic sensitivity to ragweed. Responses are determined by increased airway resistance, pulmonary immunity, and pulmonary inflammatory responses.
4. Determine if the inhalation of ultrafine carbon particles increases the airway responses of ragweed-allergic dogs during challenge with ragweed allergen.

Status: Our "susceptible" population consists of six young adult dogs born to breeders having high serum IgE levels. These dogs were immunized by multiple injections of ragweed in aluminum hydroxide starting shortly after birth. We previously showed that immunizing dogs from allergic breeders to ragweed early in life induces allergic immunity characterized by elevated eosinophils, total IgE, anti-ragweed IgE, and airway responses to both histamine and ragweed. Six age-matched dogs not previously immunized to ragweed are used as the "non-susceptible" controls.

During the past year, work under Aims 1 and 2 have been completed. Baseline lung lavage cell cytology, airway reactivity to ragweed and histamine, and antibody production are shown in Figures 2-7. To localize ragweed immune responses to the lungs, dogs in both groups were challenged three times by inhalation of ragweed. About 250 mg of ragweed was deposited in the lungs at each exposure. There was an increase in the level of total IgE and anti-ragweed IgE and IgG in serum from the ragweed sensitized dogs, while anti-ragweed antibody could not be measured in serum from control dogs (Fig. 2). Antibody levels for total IgE and anti-ragweed IgG were also elevated in the lung lavage of ragweed sensitized dogs (Fig. 3). Neutrophils were elevated in both groups, but only the dogs sensitized to ragweed as puppies had increased numbers of eosinophils in lung lavage samples (Fig. 4). These data show that inhaled ragweed induces inflammatory responses in the lungs of both ragweed sensitized and control dogs. As shown in Figures 5-7, dogs immunized with ragweed as puppies had a higher level of airway reactivity to both histamine and ragweed than the dogs not previously immunized to ragweed.

Figure 2. Serum antibody and immunoglobulin responses in allergic and non-allergic dogs following ragweed aerosol challenge; total serum IgE (A), ragweed-specific serum IgE (B), and ragweed-specific serum IgG (C). Data are presented as the mean ± standard error of the mean at different time points following exposure. Arrows indicate the time of the three inhalation exposures to ragweed.

Figure 3. Bronchoalveolar lavage fluid total IgE (A), and ragweed-specific IgG (B) in allergic and non-allergic dogs following ragweed aerosol challenge. Data are presented as the mean ± standard error of the mean at different time points following exposure. Arrows indicate the time of the second aerosol exposure.

Figure 4. Eosinophil (A) and neutrophil (B) counts in bronchoalveolar lavage fluid from allergic and non-allergic dogs following ragweed aerosol challenge. Data are presented as the mean ± standard error of the mean at different time points following exposure.

Figure 5. Kinetics of change in pulmonary resistance to histamine (5 breaths of 10mg/ml) in non-allergic and allergic dogs. Data are presented as the mean ± standard error of the mean at different time points before and after exposures.

Figure 6. Pulmonary resistance in response to increasing doses of histamine in non-allergic (A) and allergic (B) dogs before and after two inhalation exposures to ragweed. Data are presented as the mean ± standard error of the mean.

Figure 7. Pulmonary resistance in response to increasing doses of ragweed in non-allergic (A) and allergic (B) dogs before and after two inhalation exposures to ragweed. Data are presented as the mean ± standard error of the mean.

A system was developed for exposing dogs by inhalation to ultrafine carbon particles and subsequently challenging them with either ragweed or water aerosol to conduct the research under Aims 3 and 4. Ultrafine carbon particles were generated using the Palas electric arc generator and carbon electrodes. A key issue is whether the particle size remains in the ultrafine range at the high exposure concentration desired (approximately 250 µg/m3). Figure 8 shows particle size and concentration data for the exposures conducted under Aims 3 and 4. Particle size was measured using a Scanning Mobility Particle Sizer (SMPS), and mass concentration was measured by filter samples. The results demonstrate that the count median aerodynamic diameter was near 35 nm for all exposures, with a geometric standard deviation of 1.5. Thus, the system successfully exposed the dogs to ultrafine carbon at the concentration desired.

Figure 8. Summary of ultrafine particle exposure for Aims 3 (A) and 4 (B). Dogs were exposed individually to the following particles concentrations for 1 hr.

All six immunized and six non-immunized dogs were exposed by inhalation for 1 hour to ultrafine carbon particles and then challenged with either water (Aim 3) or ragweed (Aim 4). The results suggest that particle exposure caused no increase in airway reactivity during water challenge in either immunized or non-immunized dogs. Additionally, it appears that non-immunized dogs do not have an altered airway response to challenge following both particle and ragweed exposure.

Schedule for Completion: Aims 3 and 4 will be completed by the end of May 2000. The final lavage samples will be obtained on May 1st. Analysis of the samples collected under Aims 3 and 4 will be completed by the end of June 2000. The analyses will include antibody levels in lung lavage fluid and serum (IgEtotal, anti-ragweed IgE, IgG), mucin (protein) levels in lung lavage, cell differentials in lung lavage and blood, glutathione levels in lung lavage cells and fluid, and Th1 and Th2 cytokine mRNA levels in lavage cells (IFN-g, IL-2, IL-4 and IL-5).