title: Nonviral human beta defensin-3 expression in a bioengineered human skin tissue: a therapeutic alternative for infected wounds.
abstract: The innate immune system differentially regulates the expression of host defense peptides to combat infection during wound healing. We enhanced the expression of a host defense peptide, human beta defensin-3 (hBD-3), in keratinocytes to generate a three-dimensional biologic dressing to improve healing of infected wounds. The NIKS human keratinocyte cell line was stably transfected ex vivo with a construct containing an epidermis-specific promoter driving hBD-3 (NIKS(hBD)             (-3) ) using nonviral methods. Levels of hBD-3 mRNA and protein in three-dimensional skin tissue produced from NIKS(hBD)             (-3)  were determined using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Tissue architecture was characterized by hematoxylin and eosin staining and by indirect immunofluorescence using proliferation and keratinocyte differentiation markers. Antimicrobial activity was assessed using an in vitro bacterial growth assay and in vivo using a murine burn infection model. Three-dimensional full thickness skin tissues containing epidermal NIKS(hBD)             (-3)  or control NIKS possessed histologic features of interfollicular epidermis and exhibited normal tissue growth and differentiation. NIKS(hBD)             (-3)  tissue contained approximately fivefold more hBD-3 protein than tissue containing unmodified control NIKS. In vitro studies showed that NIKS(hBD)             (-3)  tissue produced a significant reduction in the growth of Staphylococcus aureus multiple peptide resistance factor (mprF) compared with control tissue. In an in vivo infected murine burn model, NIKS(hBD)             (-3)  tissue resulted in a 90% reduction in bacterial growth. These results demonstrate that sustained delivery of hBD-3 by a bioengineered skin tissue results in a therapeutically relevant reduction in growth of a S. aureus strain in an animal model of infected third-degree burn wounds.
authors.affiliation: Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
authors.forename: Angela L
authors.initials: AL
authors.lastname: Gibson
authors.forename: Christina L
authors.initials: CL
authors.lastname: Thomas-Virnig
authors.forename: John M
authors.initials: JM
authors.lastname: Centanni
authors.forename: Sandy J
authors.initials: SJ
authors.lastname: Schlosser
authors.forename: Colette E
authors.initials: CE
authors.lastname: Johnston
authors.forename: Kelly F
authors.initials: KF
authors.lastname: Van Winkle
authors.forename: Andrea
authors.initials: A
authors.lastname: Szilagyi
authors.forename: Li-Ke
authors.initials: LK
authors.lastname: He
authors.forename: Ravi
authors.initials: R
authors.lastname: Shankar
authors.forename: B Lynn
authors.initials: BL
authors.lastname: Allen-Hoffmann
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002056:Burns / Q000378:metabolism* / Q000382:microbiology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D015870:Gene Expression; D006801:Humans; D051379:Mice; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013207:Staphylococcal Skin Infections / Q000378:metabolism* / Q000382:microbiology; D013211:Staphylococcus aureus / Q000472:pathogenicity*; D014945:Wound Healing / Q000235:genetics; D014946:Wound Infection / Q000378:metabolism* / Q000382:microbiology; D023083:beta-Defensins / Q000378:metabolism*
chemical_list: C422950:DEFB103A protein, human; D012333:RNA, Messenger; D023083:beta-Defensins