Exposure to the Asp f 1 allergen in nonindustrial occupational environments

Sick Building Syndrome is a prevalent problem with patient complaints similar to typical allergy symptoms. Unlike most household allergens, the Asp f 1 allergen is conceivably ubiquitous in the work environment. This project examined levels of the Asp f 1 allergen in office and non-industrial occupational environments, and studied the bioaerosol and dust reservoirs of Aspergillus fumigatus responsible for those levels. ^ Culturable bioaerosols of total mesophilic fungi were sampled with Andersen N6 impactors. Aggressive airborne and bulk dust samples were concurrently collected and assayed for Asp f 1. Bulk dusts were selectively cultured for A. fumigatus. Samples were collected during both wet and dry climatological conditions to examine the possibility of Asp f 1 increases due to fungal growth blooms. ^ Only very low levels of Asp f 1 were detected in relatively few samples. Analysis of wet versus dry period samples showed no differences in Asp f 1 levels, although A. fumigatus counts from dusts did fluctuate significantly with exterior moisture events as did indoor prevalence of total colony forming units. These results indicate that even in the presence of elevated fungal concentrations, levels of Asp f 1 are extremely low. These levels do not correlate with climatological moisture events, despite distinct fungal blooms in the days immediately following those events. Non-industrial office buildings devoid of indoor air quality issues did not demonstrate significant levels or occurrence of Asp f 1 contamination in the geographical region of this study. ^

ALLERGEN SENSITIZATION AND IMMUNOMODULATION BY SYNTHETIC LIGANDS, PUL-042, IN AN ALLERGEN-INDUCED ASTHMA MURINE MODEL

Allergen-induced asthma is the leading form of asthma and a chronic condition worldwide. Common allergens are known to contribute to the pathogenesis of this disease. Murine models of allergic asthma have mostly used an intraperitoneal route of sensitization (not airway) to study this disease. Allergic asthma pathophysiology involves the activation of TH2-specific cells, which triggers production of IgE antibodies, the up-regulation of TH2-specific cytokines (i.e. IL-4, IL-5, IL-9 and IL-13), increased airway eosinophilia, and mucin hypersecretion. Although there are several therapeutics currently treating asthmatic patients, some of these treatments can result in drug tolerance and may be linked to increased mortality. CpG oligodeoxynucleotides (ODNs) is a synthetic ligand that targets Toll-like Receptor (TLR) 9. It has been evaluated as a therapeutic agent for the treatment of cancer, infectious diseases, and for treating allergy and asthma. PUL-042 is also a synthetic TLR ligand and is composed of two agonists against TLR2/6 heterodimer and TLR9. Previous studies have evaluated PUL-042 for its ability to confer resistance against bacterial and viral lung infection. These findings, combined with studies performed using CpG ODNs, led to speculation that PUL-042 dampens the immune response in allergen-induced asthma. My thesis research investigated airway route sensitization and airway delivery of PUL-042 to evaluate its effects in reducing an allergen-induced asthma phenotype in a murine model. The results of this study contribute to the foundation for future investigations to evaluate the efficacy of PUL-042 as a novel therapy in allergic-asthma disease.

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

Evaluation of the C5a anaphylatoxin and its receptor (CD88) in allergic lung disease

Allergic asthma is characterized by airflow obstruction, airway hyperresponsiveness (AHR) and chronic airway inflammation. We and others have reported that complement component C3 and the anaphylatoxin C3a receptor promote while C5 protects against the development of the biological and physiological hallmarks of allergic lung disease in mice. In this study, we assessed if the protective responses could be mediated by C5a, an activation-induced C5 cleavage product. Mice with ablation of the C5a receptor (C5aR) either by genetic deletion or by pharmacological blockade exhibited significantly exacerbated AHR compared to allergen-challenged wild-type (WT) mice. However, there were no significant differences in many of the other hallmarks of asthma such as airway infiltration by eosinophils or lymphocytes, pulmonary IL-4-producing cell numbers, goblet cell metaplasia, mucus secretion or total serum IgE levels. In contrast to elevated AHR, numbers of IL-5 and IL-13 producing pulmonary cells, and IL-5 and IL-13 protein levels, were significantly reduced in allergen-challenged C5aR-/- mice compared to allergen-challenged WT mice. Administration of a specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist before each allergen-challenge abolished AHR in C5aR-/- as well as in WT mice. Pretreatment with a C3aR antagonist dose-dependently reduced AHR in allergen-challenged WT and C5aR-/- mice. Additionally, allergen-induced upregulation of pulmonary C3aR expression was exaggerated in C5aR-/- mice compared to WT mice. In summary, deficiency or antagonism of C5aR in a mouse model of pulmonary allergy increased AHR, which was reversed or reduced by blockade of the cysLT1R and C3aR, respectively. In conclusion, this study suggests that C5a and C5aR mediate protection against AHR by suppressing cysLT and C3aR signaling pathways, which are known to promote AHR. This also supports important and opposing roles of complement components C3a/C3aR and C5a/C5aR in AHR. ^