Mouse salivary glands and human beta-defensin-2 as a study model for antimicrobial gene therapy: technical considerations.

Transduction of salivary glands with antimicrobial peptide genes has great potential for oral infection control. Our ultimate goal is to introduce antimicrobial peptide genes into salivary glands that secrete these peptides into saliva to control bacterial/fungal infection in the oral cavity. However, an animal study model to test this potential has not been established. Therefore, we determined to test (i) whether the potent antimicrobial peptide human beta-defensin-2 (hBD-2) can be overexpressed in saliva after transduction of salivary glands and (ii) whether oral fungal infection can be developed in a NOD/SCID murine model. Lentiviral vector SIN18cPPTRhMLV bearing hBD-2 cDNA was introduced into SCID mouse submandibular glands via cannulation. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry or enzyme-linked immunosorbent assay (ELISA) were performed to detect hBD-2 expression in glands or in saliva. Candida albicans 613p was inoculated orally into SCID mice to establish oral candidiasis. Whilst expression of hBD-2 was detected in mouse salivary glands by RT-PCR and immunohistochemistry 1 day or 1 week following delivery of lentivirus, hBD-2 was not detected in saliva. There was recoverable C. albicans from the oral cavity and gastrointestinal tract 4 days to 4 weeks after infection, but there was no establishment of observable oral candidiasis in SCID mice under a stereomicroscope. Our data indicate that lentiviral vectors transduce mouse salivary glands, but not at a sufficient level to allow hBD-2 detection in saliva. Other vectors for gene transduction and additional treatment of SCID mice to establish oral candidiasis are needed in order to utilise mouse salivary glands to test antimicrobial gene therapy.

Capacity of human beta-defensin expression in gene-transduced and cytokine-induced cells.

The purpose of this study was to determine the capacity of cells transduced with human beta-defensins (HBDs) to express antimicrobial peptides, since sufficient expression level is required for effective antimicrobial activity. Retroviral vector pBabeNeo and lentiviral vector SIN18cPPTRhMLV (SIN18) carrying HBDs were utilized to transduce non-HBD-expressing cells such as fibroblasts or HBD-producing oral epithelial cells. We found that HBD-3 gene transfer to fibroblasts was possible not via retrovirus but by direct vector transfection. SIN18 had high transduction efficiencies (80.9-99.9%) and transduced cells expressed higher amounts of HBD-2 than those by pBabeNeo. Primary human gingival epithelial cells (HGECs) expressed greater amounts of HBD-2 than primary fibroblasts after lentiviral transduction. Additionally, HBD-2 secretion from transduced HGECs cells was further increased when stimulated with IL-1 or TNFalpha. Our data indicate that while HBD-2 expression is limited in primary fibroblasts, its expression in HGECs may be maximized by gene transduction plus cytokine induction.