Moreover, proteolytic HDM allergens (including group 1 allergens and serine peptidase) not only compromise the epithelial barrier but also activate multiple innate immune signaling pathways. This activation triggers the release of proinflammatory cytokines and chemokines, such as interleukin-8 (IL-8) and thymic stromal lymphopoietin (TSLP), which further promote a Th2-skewed immune response [18; 25; 26; 27]. However, relatively few studies have comprehensively evaluated the combined impact of these allergens. Existing studies indicate that dual or multiple exposure may exacerbate respiratory pathology beyond the sum of individual effects, a hypothesis that could explain observed increases in asthma prevalence and severity in individuals exposed and sensitized to both HDM and ragweed allergens [23]. The synergistic effects of ragweed and HDMs may result in compounded inflammatory responses, heightened epithelial permeability and increased risk of chronic respiratory dysfunction [9; 10; 28]. This reinforces the importance of studying allergen combinations rather than isolated exposures, as real-world environmental conditions often involve concurrent allergen exposure, which may accelerate disease progression. Emerging research highlights that while each allergen alone has significant effects on the respiratory epithelium, their combination can amplify these disruptions, potentially leading to more severe consequences. This study investigates these effects on NHBE cells cultured at the air-liquid interface (ALI) in a Transwell system, which mimics the bronchial epithelium in vivo and allows for realistic allergen exposure conditions. The transepithelial electrical resistance (TEER) assay is widely utilized to assess the functionality of epithelial cell culture models grown on a permeable membrane and to provide a quantitative evaluation of paracellular permeability and tight junction integrity [29; 30]. In the respiratory tract, the epithelial cells separate inhaled air from underlying tissues and regulate ion and macromolecule transport [31; 32]. Respiratory barrier dysfunction is common in various respiratory diseases and can be triggered by exposure to a wide range of inhaled chemicals, particulates and pathogens [21; 33; 34]. TEER measures the electrical resistance across one or more cell layers grown on a porous membrane. This resistance, reflecting impedance to current flow, serves as a quantitative indicator of monolayer integrity by assessing ionic conductance. A small electrical current is applied between two electrodes placed on opposite sides of the barrier barrier--more precisely, one in the apical chamber (above the epithelial layer) and the other in the basolateral chamber of the Transwell system (below the epithelial layer)--and the voltage is recorded. The recorded TEER values correlate with barrier integrity; higher values indicate tighter cellular junctions, whereas lower values suggest increased permeability and barrier disruption. As a key measure of barrier integrity, TEER assay results can qualitatively correlate with in vivo indicators of barrier dysfunction following respiratory injury. Complementing these measurements, immunofluorescence (IF) microscopy with ZO-1 staining provides a comprehensive understanding of allergen-induced epithelial changes and a high-resolution view of epithelial architecture. ZO-1, a critical tight junction protein, plays a pivotal role in maintaining epithelial barrier function and its localization can be directly correlated with cell junction integrity. By staining ZO-1 in green and cell nuclei in blue, this study visualizes the extent to which ragweed and HDM allergens--individually and combined--disrupt the epithelial barrier at the structural level [23; 35]. Changes in ZO-1 distribution, captured through IF microscopy, are expected to align with fluctuations observed in TEER readings, thereby providing a comprehensive view of allergen impact on both functional and structural aspects of the epithelial barrier.