## 3 Results
### Revealing the Impact of Allergen Exposure on Epithelial Barrier Integrity Through TEER Monitoring
Although no significant differences were observed between the TEER values in cells exposed to either allergen extract preparation at 200 \(\upmu\)g/mL, comparative analysis revealed that single-allergen exposures (RW 200, HDM 200) caused less barrier disruption than the  
Figure 3: Changes in transpepithelial electrical resistance in response to different allergens at varying concentrations and combinations. TEER values are normalized to pre-exposure baseline levels and presented as a percentage over time (h) post-exposure. (**A**) TEER changes in response to different concentrations of HR (HR 25, HR 50 and HR 100) compared to the control group (CTR). (**B**) TEER responses to AM 100, DAM 50 and DAM 100 treatments relative to CTR. (**C**) TEER dynamics following exposure to HR 200, HDM 200 and RW 200 compared to CTR. The groups are labeled as follows: CTR: control; RW 200: RW extract at 200 \(\upmu\)g/mL; HDM 200: HDM extract at 200 \(\upmu\)g/mL; HR 200: RW+HDM extracts in equal parts at 200 \(\upmu\)g/mL; HR 100: RW+HDM extracts at 100 \(\upmu\)g/mL; HR 50: RW+HDM at 50 \(\upmu\)g/mL; HR 25: RW+HDM at 25 \(\upmu\)g/mL; DAM 100: Der p 1+Amb a 1+Amb a 11+Amb a 12 preparation corresponding to 100 \(\upmu\)g/mL of allergen extract; DAM 50: Der p 1+Amb a 1+Amb a 11+Amb a 12 preparation corresponding to 50 \(\upmu\)g/mL of allergen extract; AM 100: Amb a 1+Amb a 11+Amb a 12 preparation corresponding to 100 \(\upmu\)g/mL of allergen extract. The results are expressed as the mean \(\pm\) SD for each time point. *\(p\) < 0.01 and *\(p\) < 0.0001 versus control. combination of RW and HDM (HR 200). This supports the hypothesis that co-exposure exacerbates epithelial permeability loss. Moreover, the specific allergic preparations (DAM 100 and DAM 50 groups) demonstrated that the loss of barrier integrity is not solely concentration-dependent but is also influenced by the specific allergenic molecules within the allergen extracts. These findings point out the differential effects of allergen exposure on bronchial epithelial integrity and emphasize the utility of TEER as a quantitative measure of barrier function disruption in allergic airway diseases. Visualization of Tight Junction Integrity Through ZO-1 Immunofluorescence Imaging Following Allergen Exposure  
Immunofluorescence imaging was utilized to further assess the morphological alterations in bronchial epithelial cells following allergen exposure, with a focus on ZO-1 tight junction (TJ) proteins. Normal human bronchial epithelial cells, cultured at the air-liquid interface in a Transwell system to form a pseudostratified, ciliated epithelium, were exposed to various allergen formulations at different concentrations. Fixed tissue sections were stained for ZO-1 (green) and the cellular morphology was examined under fluorescence microscopy at 60\(\times\) magnification, as shown in Figures 4-6. Immunofluorescence analysis of ZO-1 expression in NHBE cells reveals distinct patterns of tight junction integrity across different allergen exposures, as exposed in Table 1. In the control group, ZO-1 staining appears well defined and continuous along cell borders, indicating intact epithelial barrier function. In contrast, exposure to HR at increasing concentrations (50, 100 and 200 \(\upmu\)g/mL) results in a dose-dependent disruption of ZO-1, with higher concentrations leading to fragmented staining and reduced fluorescence intensity. HR 100 (100 \(\upmu\)g/mL) exhibited pronounced morphological changes. Exposure to the DAM 100 allergenic preparation containing Der p 1, Amb a 1, Amb a 11 and Amb a 12 at 100 \(\upmu\)g/mL and HR 50 (50 \(\upmu\)g/mL) resulted in a moderate reduction in ZO-1 expression, therefore reflecting partial loss of tight junctions and showing the potential role of Der p 1 upon cell barrier alteration. The DAM 50 condition (50 \(\upmu\)g/mL) and AM 100 (100 \(\upmu\)g/mL) induced less pronounced barrier dysfunction concerning ZO-1 expression, highlighting the concentration-dependent effects of specific allergenic components on epithelial barrier integrity.