Since the discovery of natural antimicrobial peptides/proteins, researchers have tried not just to understand how these peptides/proteins work and applied them in topical use for infection control, but have also explored the possibility of linking their use to gene-based therapeutics, i.e. transducing cells with these peptide genes to augment the innate resistance of tissues/organs against infection [1
]. Tracheal antimicrobial peptide (TAP), a member of the β-defensin family of antibiotic peptides found in the tracheal mucosa of cows, was used to generate transgenic animals to produce TAP in milk that is antimicrobial [3
]. Among a long list of naturally occurring antimicrobial peptides, some well characterised ones are encoded by single genes and their products do not need post-translational processing, making them good candidates for antimicrobial gene therapy. Human β-defensins (hBDs or DEFB) have drawn considerable attention for this purpose as they have potent antimicrobial abilities and some also exert inhibitory effects on viral infections [6
]. Although only four hBDs (hBD-1–4) have been well characterised, up to 28 hBDs may exist based on genomic surveys using a computational search strategy [14
]. Fourteen hBD or DEFB gene transcripts (DEFB-1, -4 and -103–114) were investigated using reverse transcription polymerase chain reaction (RT-PCR) to detect their expression in gingival keratinocytes [16
]. Transduction of epithelial cells in skin or oral mucosa with hBDs has been tested for its potential to enhance infection control [8
]. Primary fibroblasts have also been proposed as target cells to express hBDs, but the amount of antimicrobial peptide secreted may be insufficient to exert an effect [17
Salivary glands have been considered an excellent target organ for gene-based therapeutics compared with other tissues or organs (e.g. liver and skeletal muscle) both for systemic and upper gastrointestinal tract gene therapeutic applications. Salivary glands are well encapsulated, which limits undesired extraglandular vector dissemination. In addition to being an exocrine secretion organ, physiological existence of endocrine secretory pathways in these tissues has been reported [19
]. In 1996, O’Connell et al. [2
] first utilised salivary glands as a target organ for antimicrobial gene-based therapy to control oral infection. The gene encoding the anticandidal protein histatin 3 was transferred to rat salivary glands using an adenovirus-directed approach, and up to 1 mg/mL of histatin exerting a strong candicidal effect was detected in saliva. However, the study did not test whether the secreted histatin 3 in saliva controls oral fungal infection in vivo.
hBDs are expressed in salivary glands and are detected in saliva [22
]. They are presumably among a plethora of defence mechanisms in the oral cavity to combat infections. hBD-2 is a potent antimicrobial peptide effective against a broad spectrum of bacteria, including Gram-positive bacteria, Gram-negative bacteria and fungi. Among these microbes, Streptococcus mutans
and Lactobacillus acidophilus
, which are associated with dental caries, and certain strains of periodontal bacteria such as Actinobacillus actinomycetemcomitans
and Porphyromonas gingivalis
have been shown to be sensitive to hBD-2 [18
]. hBD2 is chemotactic for T-cells, immature dendritic cells, mast cells and tumour necrosis factor-alpha (TNF-α)-primed neutrophils [10
]. Its expression in saliva may enhance not only the innate but also adaptive immunity of the oral mucosa. Together with its ability to inhibit human immunodeficiency virus (HIV) infectibility [11
], these characteristics make hBD-2 an ideal candidate for a study model of antimicrobial gene therapy to control oral infection.
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, which secrete these peptides into saliva to control bacterial/fungal/viral infection in the oral cavity. However, an animal study model to test this potential has not yet been established. In the present study, our objective was to address two questions before establishing this model: (i) whether the potent hBD-2 can be expressed in saliva after transduction of salivary glands using lentiviral vectors; and (ii) whether oral fungal infection can be developed in a NOD/SCID murine model. Our findings provide an insightful understanding on the technical issues and concerns for future investigation regarding salivary gland antimicrobial gene therapy for oral infection control.