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

Abstract

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 β-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.

Salivary Defensins and Their Importance in Oral Health and Disease

“…However, there have been no approved clinical trials involving salivary gland gene transfer. Recently, the gene transfer of hBD2 to mouse salivary glands using lentiviral vectors was attempted [111]. Although expression of hBD2 was detected in the salivary glands by RT-PCR and immunohistochemistry, it was not detected in saliva.…”

The increasing of beta-defensin-2 level in saliva after probiotic Lactobacillus reuteri administration

“…Therefore, in the salivary examination of BD-2 level by using elisa method, it is known that the mean concentration of BD-2 in the positive control group was the lowest one, about 9.50 ng/ ml, but this concentration can already be active because defensins can be active at the concentration of 1 to 10 ng/ ml. 19 Based on Table 2, the average level of BD-2 in the negative control group was different from that in the treatment group 2 (p = 0.024). In other words, it is known that the average level of BD-2 in the negative control group was significantly increased compared to that in the treatment groups 2, from 11:14 ng/ mL to 14.96 ng/ mL.…”

Specific Binding and Chemotactic Activity of mBD4 and Its Functional Orthologue hBD2 to CCR6-expressing Cells

“…In addition, correct folding is often a problem in polypeptides with a higher number of cysteine residues (20) and is achieved more readily using insect than bacterial cells. Although the antimicrobial activity of hBD2 either synthesized or produced in E. coli has been extensively tested using various Gram-positive and Gram-negative bacterial strains, fungi, and viruses (21)(22)(23), little is known about the antimicrobial activity of its mouse orthologue mBD4. The recombinant mBD4:Ig and hBD2:Ig fusion proteins both retained potent bactericidal activity against both Gram-positive and Gram-negative bacteria at physiological salt concentrations.…”