Introduction
The systemic analysis of genetic variants in their functional context will allow to finally understand the contribution of genetic predisposition to the development of bronchial astma and other atopic disorders. To reach this goal, a close interaction between population-based studies and functional analysis in animal models has to be accomplished. In this context, the utilization of an animal model allows the systematic analysis of gene-phenotype interactions and differential gene expression in the course of disease development and progression in a genetically homogeneous population and with full control of enviremental influences. The aim of this project is to increase the understanding of disease mechanisms by identifying new genes and functional relevant polymorphisms for disease development utilizing a murine model of bronchial asthma. The integration of the murine model in the concept of genetic analysis of bronchial asthma will allow us to extend observations from studies in human tissues to organs or cells that are not available in patients, to identify common mechanisms of disease development, to establish chronic disease models that closer resemble the features of allergic disease in patients, to identify new genes and relevant polymorphisms involved in the development of bronchial asthma, to functionally evaluate the role of newly identified biomarkers/ genes / polymorphisms from human and murine studies for disease development and progression, and to finally establish novel strategies for better prediction, prevention and treatment of bronchial asthma.

Project Status
Murine model of bronchial asthma
We have established a murine model of allergen-mediated airway disease that offers the unique opportunity to study in vivo allergen sensitization in a genetically homogenous population with a clearly defined disease phenotype and with full control of environmental influences.This model comprises the main feature of the disease: (I) increased production of allergen-specific IgE following systemic or inhalative sensitization with allergen; (II) marked inflammatory responses in lung tissues, and (III) development of unspecific airway hyperreactivity (AHR) detectable by in vivo lung function measurements, following allergen airway challenges of sensitized mice. We have extensively studied immunological mechanisms and environmental conditions leading to disease onset and progression and identified crucial factors for the development of murine bronchial asthma (1-10).

We were able to:
-Identify more than 1000 differentially expressed gens during the progression of allergen-mediated airway disease.
-Identify gene expression profiles explicit for specific compartments of disease manifestation: spleen, local lymph nodes, lung tissues, neuronal ganglia.
-Idenify gene expression profiles specific for certain pathophyiological aspects: allergic sensitization, airway inflammation, development of AHR.
-Identify novel genes with functional relevance for the development of allergen-induced airway inflammation (11,12).

Functional evaluation of novel biomarkers
One of the most significant benefits of using a murine model is that it allows a direct functional evaluation of novel genes and biomarkers. A good example for successful identification of a new biomarker with functional importance for disease development utilizing our approach is the signal- transducer and activator-of-transcription factor (STAT-1) (12). STAT-1 expression by epithelial cells in lung tissues of sensitized and challenged mice is upregulated and may be critical for the development of inflammatory responses and AHR following airway allergen challenges of previously sensitized mice. For functional analysis, we utilized a novel approach to inhibit STAT-1-mediated gene expression by applying a decoy technique.

This approach resulted in:
-Attenuated leukocytic airway inflammation
-Decreased Th2-cytokine production in lung tissues
-Reduced expression of adhesion molecule VCAM-1 by endothelial cells and of costimulated antigen CD40 by epithelial cells
-Inhibtion of the development of in vivo AHR STAT-1 ( and other newly identified factors with functional properties for disease development, like inducible co-stimulator, ICOS) may thus serve as a novel diagnostic or therapeutic target for disease prediction and/or prevention.

Results
Early prediction and effective prevention of allergic diseases requires a better understanding of the genetic program underlying the pathophysiological mechanisms. In order to evaluate the molecular changes initiated by allergen sensitization and airway challenges in a murine model of bronchial asthma , gene expression profiles were analyzed by microarray techniques (Fig 1+2) in two different mouse strains (BALB/c, C57/Bl6) and four different organs (lung, spleen, lymph nodes and ganglia), which are involved in the development of the allergic phenotype. Comparative analysis of expression profiles of lung tissues in the early phase of airway inflammation following allergen sensitization and airway challenges revealed that a total of 1,1% of all genes were upregulated compared to sensitized and sham-challenged animals. Two of these genes had so far been described in oxidative stress responses, yet, their particular role in the development of allergen-induced airway inflammation remains indecisive. Therefore, we further delineated the involvement of the novel genes and confirmed the microarray results via quantitative PCR (Fig 3+4). Western blot analysis and immunhistology showed that RNA-upregulation translated into enhanced protein production and distinct expression patterns in the bronchial epithelium for both genes. The data suggest that these genes represent novel and common inflammatory genes involved in early stages during the development of allergen mediated airway disease. Consequently, these genes are currently tested for their functional relevance for the occurence of allergic asthma and may ultimately provide new targets for the diagnosis or treatment of the disease.

Fig 1: RNA expression profiles of lung tissue was examined by using a murine Affymetrix  chip (U74 A), which represents ~12000 already annotated mouse genes.

Fig 2: Scatter plot of expressed genes, comparing naïve mice and animals with allergen induced airway inflammation.

Fig 3: Qualitative verification by PCR

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