In the pathogenesis of periodontal disease, invasion of the gingival epithelium by putative pathogens appears to be critical. While many mechanisms exist to protect the mucosal surface against colonization, it is unclear if gingival epithelial cells can defend against invading bacteria. Gingival keratinocytes do express calprotectin, a cytoplasmic antibacterial protein complex. the Principal Investigator hypothesizes that calprotectin serves as an innate intraepithelial antimicrobial mechanism. To test this novel hypothesis, calprotectin will be expressed and purified and complexes will be used to determine the subunit requirements for antimicrobial activity. Normal human gingival keratinocytes will be screened for expression of calprotectin. Calprotectin-negative and calprotectin-positive normal gingival keratinocytes will be compared for resistance to colonization and invasion by putative periodontal pathogens, including Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. To show that the antimicrobial effect is restricted to calprotectin, HeLa cells will be stably transfected with calprotectin-specific cDNA and compared to control negative transfections for resistance to colonization and invasion. To demonstrate the change in innate antimicrobial activity, immortalized keratinocyte cell lines will be screened for expression of calprotectin. Specific expression of calprotectin in selected immortalized lines and normal gingival keratinocytes will be abrogated with antisense DNA and cells will then be tested for altered antimicrobial properties. These studies will define if calprotectin serves an innate defense against bacterial invasion in vitro and provide strong evidence for a fundamental role in resistance to infections.
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