At present, there are few methods to selectively transfer genes to non-dividing airway smooth muscle cells. This is a major problem in the development of gene therapy approaches to treat the airway hyperresponsiveness and remodeling associated with asthma. We have identified a DNA sequence that increases nuclear localization and subsequent gene expression uniquely in smooth muscle cells, a critical target in asthma gene therapy. We hypothesize that the cell-selective nuclear import of the smooth muscle gamma actin (SMGA) promoter is mediated by the transcription factors SRF and Nkx3.1/3.2 that are expressed in smooth muscle cells but not other cells of the airway. With our development of a new method for highly efficient gene delivery to the lungs and airways of living animals using electroporation, we are in a unique position to test the effects of this cell-selective nuclear import sequence on smooth muscle transfection in animal models for asthma. We hypothesize that the SMGA DNA nuclear targeting sequence will lead to gene transfer and expression only in airway smooth muscle cells, and not in airway epithelial or other lung cells of living animals. This proposal is designed to test this hypothesis and will led to the creation of new airway gene therapy vectors that are both cell-specific and capable of greater gene transfer efficiencies. Finally, we will use the information gained to test the efficacy of these vectors in an animal model for airway hyperresponsiveness using PKC and MLCK dominant-negative mutants and MLCP gene transfer.

Polymorphisms that are associated with increased risk for asthma have been identified. However, a causative role for these alleles in the pathogenesis of asthma has not been established. The goal of this project is to develop mouse models for testing for causality of human genetic variants in functional screens of allergic airway disease. We suggest that these animals will advance this field of study by providing: 1.) a mechanism for direct in vivo testing of the propensity for alleles present in the human population to promote allergic airway disease and other inflammatory diseases. 2.) mouse models of asthma that more closely resemble the human disease. 3.) a means to identify novel targets for the treatment of asthma. 4.) "humanized" mouse models for direct testing of pharmacological reagents that will be applicable to humans. Two strategies will be used to generate these mouse lines. In the first, amino acids present in the mouse gene are changed in situ, to those associated with asthma in the human population. In the second, the entire mouse gene is deleted and replaced with the corresponding human sequences. While a number of loci conferring susceptibility to asthma and atopy have been identified, some of the most compelling data implicates the genes for IL-4, IL-13, and their respective receptors. We will therefore begin with the humanization of these genes and testing of normal and disease associated alleles in mouse models.

Engagement of cell surface receptors results in the activation of numerous biochemical second messenger cascades. These signaling pathways must be integrated as the appropriate cellular response is programmed. Recent evidence from numerous cell types has demonstrated the critical role played by adapters, proteins with modular domains which mediate intermolecular interactions, in the creation of multimolecular complexes essential for coordinating signaling events. Mast cells, critical effectors in the initiation and propagation of airway hyperreactivity, express adapter molecules which are known to play important roles in the regulation of signal transduction cascades. This project will investigate the mechanism by which two of these adapters, SH2 domain containing leukocyte phosphoprotein of 76 kDa (SLP-76) and adhesion and degranulation promoting adapter protein (ADAP), regulate receptor mediated activation of mast cells. We have initiated experiments to assess the structural features of SLP-76 and ADAP important for function in the primary mast cells ex vivo by measuring the role of these proteins on signaling events downstream of the high affinity receptor for IgE and evaluating how these adapters integrate signals important for integrin receptor function. Experiments are also described to generate genetically altered mice with targeted mutations in these proteins to examine the effect of these manipulations on mast cell function in vivo and how this will impact murine models of asthma. Collectively, we anticipate that our studies will provide new insights into the roles these adapter proteins play in mast cell function and will hopefully provide clues to potential avenues for novel asthma therapies.

Asthma is a significant cause of morbidity and mortality for African Americans and asthma is increasing in prevalence. Both environmental and genetic factors contribute to the pathogenesis of asthma. It is our hypothesis that genetic regulation of endogenous nitric oxide (NO) production, in the setting of environmental stimuli such as endotoxin, contributes to the pathogenesis of asthma in humans. Asthma, airway hyperresponsiveness, and atopy are all associated with increased exhaled NO levels. Importantly, NO has several proinflammatory activities that contribute to the pathogenesis of asthma and other diseases associated with chronic inflammation. The production of NO in normal individuals and in individuals with asthma is mediated by bronchial epithelial cells that express the enzyme inducible nitric oxide sythase (NOS2). Lipopolysaccharide (LPS) is a form of endotoxin that is an important inducer of NOS2 expression in human cells. Therefore, we believe that environmental exposure to LPS is associated with production of pathogenic levels of NO in individuals with the appropriate genetic background. To date, we have identified three single nucleotide polymorphisms (SNP) in Africans with malaria that are associated with increases in systemic NO production. Therefore, to test our hypothesis, we will identify functional SNPs in the NOS2 gene that are associated with either the basal or LPS-induced overproduction of exhaled NO in healthy African Americans. These studies will establish NO as a treatment target in asthma, identify the relationship of environmental exposure to LPS on exhaled NO levels and identify genetic markers that may be used to target preventative strategies and predict therapeutic responses for patients with asthma

Mast cells play a crucial role in the allergic airway response of asthma via the high-affinity receptor for IgE, FceRI. The recruitment and activation of mast cells is orchestrated by T cells, which are also involved in pathogenesis. The transmembrane adapter protein LAT is essential for FceRI-mediated signaling in mast cells and for T cell activation following TCR ligation. LAT is believed to direct the assembly of a multiprotein signaling complex comprising the adapter molecules Gads and SLP-76, and the enzyme phospholipase Cg1 (PLCg1). While rapid progress is being made towards the identification of LAT-associated proteins, and on understanding their role in lymphocyte development and function, much less is known about the biochemical properties and three-dimensional structure of LAT-nucleated signaling complexes. We propose to study the assembly and structure of the postulated LAT/Gads/SLP-76/PLCg1 complex using a multidisciplinary approach combining biophysical methods to measure thermodynamic binding parameters with X-ray crystallography to determine three-dimensional structures. First, we will produce full-length and truncated versions of LAT, Gads, SLP-76 and PLCg1 in milligram amounts using bacterial or insect cell expression systems. Second, we will assess the role of cooperative binding interactions in assembly of the LAT/Gads/SLP-76/PLCg1 complex. Third, we will reconstitute binary, and higher-order (ternary and quarternary), LAT-based complexes in vitro for the purpose of visualizing specific protein-protein interfaces by X-ray crystallography. These studies will significantly advance our knowledge of the molecular basis for LAT-mediated signal transduction. Moreover, they will contribute to the development of structure-based therapies for the treatment of asthma based on specific blockade of mast cell or T cell activation using small molecules to disrupt the assembly of LAT-nucleated signaling complexes.

Asthma is a chronic inflammatory disorder of the airways. The contribution of Th2-mediated adaptive immune responses including the recruitment and activation of mast cells and eosinophils is well established in the pathogenesis of asthma, yet almost nothing is known about the underlying mechanisms that trigger allergic inflammation. Mammalian Toll-like receptors (TLRs) have recently been established as critical signal transducing receptors that initiate innate immune and inflammatory responses. The overall objective of this proposal is to investigate a possible role of TLRs in the pathogenesis of allergic inflammation. The airway epithelium is continuously exposed and challenged by a variety of environmental stimuli, including pathogens and pathogen-derived products known to trigger TLRs. Stimulation of TLRs leads to the activation of the NF-kB signaling pathway that plays a crucial role in allergic and other types of inflammation, in part due to the induction of chemokines and cytokines that mediate Th2 effector responses. We have generated two types of transgenic mice that express constitutively active TLR4 under control of inducible promoters. In one transgenic line, the expression of constitutively active TLR4 is restricted to the airway epithelium, while in the other line, this expression is restricted to dendritic cells. These mice will be used to study the role of TLRs in the initiation of allergic inflammation and to evaluate the contribution of the TLR pathway to the negative regulation of Th2 responses. In addition, we will analyze the induction of allergic inflammation in mice that are genetically deficient for MyD88, a critical downstream component of the TLR signaling pathway.

CD4 T cell responses to fungal antigens can result in allergic asthma. Spores (conidia) from the ubiquitous fungus Aspergillus fumigatus are commonly inhaled and, in a subset of individuals, prime Th2 CD4 T cell responses that lead to increased pulmonary mucus secretion, peribronchial fibrosis, eosinophil recruitment and IgE synthesis. The experiments described in this grant proposal will investigate the early, in vivo events that result in the activation and differentiation of Aspergillus fumigatus specific CD4 T cells following inhalation of conidia. It is our hypothesis that conidia and associated fungal molecules induce inflammatory processes that sway the subsequent CD4 T cell response. Furthermore, we hypothesize that the particulate nature of Aspergillus fumigatus conidia contributes to the priming and differentiation of CD4 T cells, distinguishing our proposed model from existing ones investigating T cell responses to inhaled soluble antigens. Our first aim is to generate and characterize CD4 T cell lines and hybridomas from mice immunized with Aspergillus fumigatus antigens and conidia. Our second aim is to clone T cell receptor (TCR) genes from Aspergillus fumigatus specific T cells clones and generate TCR transgenic mouse strains. In the third aim we describe adoptive transfer studies that characterize the trafficking, activation, proliferation and differentiation of Aspergillus fumigatus specific CD4 T cells during the early stages of allergic asthma pathogenesis. Generation of TCR transgenic mice specific for fungal antigens is essential for the in vivo analysis of the early events leading to inflammation and pulmonary remodeling of asthma. Defining the connections between inflammatory responses to particulate fungal antigens and Th2 CD4 T cell responses to Aspergillus fumigatus conidia may result in novel therapeutic strategies to treat and possibly prevent allergic asthma.

The active principle in marijuana, D9-tetrahydrocannabinol (D9-THC), dilates bronchial smooth muscle in humans, suggesting that cannabis-like (cannabinoid) compounds may be applied for the treatment of airway diseases. The therapeutic significance of this finding is obscured, however, by our limited understanding of the roles played by cannabinoid receptors and their endogenous ligand, anandamide, in airway physiology. We have recently shown that anandamide is produced in rodent lungs. We have also found that this compound exerts multiple effects on bronchial responsiveness, which are mediated by activation of CB1 cannabinoid receptors in the airways. Based on these findings, we hypothesize that anandamide participates in the pathophysiological control of bronchial responsiveness. Our experiments will address four questions that are pertinent to a test of this hypothesis.

1. What are the receptor mechanisms responsible for anandamide release in the lungs? We will use a perfused lung model to determine whether neurotransmitters and inflammatory mediators stimulate anandamide release.
2. What are the mechanisms responsible for anandamide inactivation in the lungs? In the same model, we will also examine the pathways of anandamide clearance.
3. Does anandamide affect neurotransmitter function in the lungs? Additional in vitro models will be utilized to study the effects of anandamide on (a) neurotransmitter release and (b) contractile responses to neurotransmitters and inflammatory mediators.
4. Is anandamide involved in allergic asthma? We will address this question by using in vivo models, including CB1 receptor knock-out mice.

These studies may provide a framework to the development of more selective cannabinoid-based agents for the treatment of respiratory pathologies.

Allergic asthma is a complex disorder characterized by local and systemic allergic inflammation and reversible airway obstruction. Interleukin-13 is a central mediator of allergic asthma; it is produced by Th2 cells and mast cells, and its levels are increased in airway tissues of human asthmatics and animal models of asthma. In Th2 cells as well as mast cells, IL-13 production is controlled by the calcium-regulated transcription factor NFAT. NFAT consists of four family members, at least three of which (NFAT1 (p, c2), NFAT2 (c, c1), NFAT4 (x, c3)) are expressed in both cell types. Strong but circumstantial evidence indicates that the different NFAT family members exhibit cell-type- and gene-specific differences in their ability to regulate gene transcription in activated immune cells. Of specific relevance to this proposal, we have shown that NFAT1, but not NFAT2, is essential for IL-13 transcription by mast cells. This is very different from the situation in Th2 cells, where both family members contribute to IL-13 expression. Here we propose to investigate the molecular mechanisms underlying the differential regulation of the IL-13 gene by these two NFAT proteins in mast cells, and to determine whether these two NFAT family members have distinct effects on asthma induction and development in an in vivo model of allergen-induced asthma in mice.

NF-kB is composed of either heterodimeric subunits (e.g. p50-p65 heterodimers) or homodimeric subunits (e.g. p50 homodimers). In this study we are interested to determine the role of the subset of genes controlled by the p50 homodimers, and regulated by a novel derepression mechanism, to the pathogenesis of asthma. p50 homodimers are detected in the nucleus and are able to function as repressors. In this study we propose to focus on increasing our understanding of the genes repressed by p50 homodimers as we hypothesize that derepression of these genes in asthma may play an important role in epithelial repair and airway remodeling. Cytokine stimulation results in derepression of a specific subset of p50 homodimer regulated genes, including the epithelial-expressed gene KAI-1, which encodes a tetraspanin, and may play an important role in airway remodeling as it associates with the Epidermal Growth Factor (EGF) receptor and attenuates EGF receptor signaling. As p50 homodimers induce expression of KAI-1, and KAI-1 attenuates epithelial EGF receptor signaling and function, p50 homodimers could attenuate EGF receptor signaling and reduce epithelial airway repair in asthma. In addition, levels of TGF-beta released by airway epithelial cells are significantly increased if repair is retarded, suggesting that if KAI-1 inhibited EGF receptor signaling increased levels of TGF-b could result and lead to enhanced peribronchial fibrosis. Therefore in this study we propose to investigate the role of p50 homodimers in the repression of genes important to the pathogenesis of asthma, focusing on KAI-1, and identifying "novel" genes regulated by p50 homodimers and determining their role in asthma.