Objective: The goal of this study was to confirm that epithelial KB cells provide an environment that modulate bacterial gene expression and produce patterns similar to those discovered in humans by In Vivo Induced Antigen Technology (IVIAT). Methods: 1.0x105 KB cells were infected with in vitro-grown Aa at a multiplicity of infection of 1000:1 Aa to KB, respectively. Aa was allowed to invade for two hours at 37°C in 10% carbon dioxide. Controls were kept from invading by lysing the KB cells with Triton X-100 prior to infection. After infection, extracellular Aa was inactivated by treatment with gentamicin. Intracellular bacteria were then recovered by lysing the infected KB cells. Bacterial counts were performed to determine the efficiency of the infection. Total RNA was extracted, purified, and quantified using standard procedures. Reverse transcription was performed to generate complementary-DNA. This cDNA and relevant controls were used as templates for quantitative real-time polymerase chain reaction (PCR). The cDNA templates were tested alongside their RNA counterparts using specific primers. The intracellular Aa gene expression was quantified using standard curves and different algorithms and compared to the gene expression levels of Aa grown extracellularly. The gene expression differences were analyzed using a Student T-test and was considered statistically relevant at p<0.005. Results: Every IVIAT gene tested by this method was found to have significant intracellular induction as compared to extracellularly-grown bacteria. Conclusion: Our data support that epithelial KB cells are an appropriate model for Aa infections and can be used to measure absolute gene expression quantitatively and in vivo. Based on these findings, we suggest that KB cells are a suitable, convenient, and reproducible cell line to study the function in pathogenesis of these genes. This research was supported by a UFCD Student Research Program Fellowship, the Center for Molecular Microbiology and NIH/NIDCR RO1 DE13523.

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