## 3 Results
### Revealing the Impact of Allergen Exposure on Epithelial Barrier Integrity Through TEER Monitoring
Following allergen exposure, ZO-1 immunofluorescence imaging revealed significant alterations in epithelial morphology that correlated with the TEER measurements. In the RW 200 (200 \(\upmu\)g/mL) condition, ZO-1 staining exhibited considerable disruption of the tight junction (TJ) structures, reflecting moderate to severe structural changes in epithelial integrity, even though they seem less severe than high-dose HR or HDM exposure. In contrast, HDM 200 (200 \(\upmu\)g/mL) exposure induced more pronounced changes in the epithelial architecture, causing a marked loss of ZO-1 expression and significant tight junction impairment. This suggests that HDM exposure has a stronger impact on epithelial integrity compared to RW alone, as confirmed by both the imaging and TEER results. The combined HR 200 condition (RW + HDM at 200 \(\upmu\)g/mL) produced the most significant morphological disruption, with ZO-1 staining exhibiting extensive disorganization of tight junctions. The TEER measurements further validated the enhanced epithelial permeability observed in the imaging, supporting the hypothesis that co-exposure to RW and HDM allergens exacerbates epithelial barrier dysfunction. Figure 4: Immunofluorescence staining of zonula occludens-1 of normal human bronchial epithelial cell cultures. Cells were exposed to different allergen extracts and allergen molecule combinations to evaluate TJ alterations induced by allergen exposure. Fixed tissue sections were stained for ZO-1 (green), with nuclei counterstained using DAPI (blue) and imaged by fluorescence microscopy. Scale: 50 \(\upmu\)m. The groups are labeled as follows: CTR: control; RW 200: RW extract 200 \(\upmu\)g/mL; HDM 200: HDM extract 200 \(\upmu\)g/mL; HR 200: RW + HDM extracts in equal parts at 200 \(\upmu\)g/mL; HR 100: RW+HDM extracts 100 \(\upmu\)g/mL; HR 50: RW+HDM 50 \(\upmu\)g/mL; HR 25: RW+HDM 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. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Exposure Condition** & \
\begin{tabular}{c} **Changes in** \\ **Normalized** \\ **TER (\%) at 72 h** \\ \end{tabular} &
\begin{tabular}{c} **Tight Junction Integrity** \\ **(ZO-1 IF Staining \({}^{*}\))** \\ \end{tabular} & **Implications for Airway Health** \\ \hline Control (no allergen) & 105.85\% \(\pm\) 6.19\% & 0: Intact ZO-1 & Normal epithelial integrity \\ \hline \hline \end{tabular}
\end{table} 
Table 1: A summary of the TEER assessment, tight junction integrity and airway health implications under various experimental conditions. This table presents the effects of various exposure conditions on transepithelial electrical resistance, tight junction integrity (evaluated through ZO-1 immunofluorescence staining) and their potential implications for airway health. TEER values (changes in normalized TEER at 72 h) indicate the barrier integrity of epithelial cells, while ZO-1 staining provides a visual assessment of tight junction organization. The findings help illustrate how different exposures influence epithelial barrier function, with potential consequences for respiratory health.