Local Burn Injury Promotes Defects in the Epidermal Lipid and Antimicrobial Peptide Barriers in Human Autograft Skin and Burn Margin: Implications for Burn Wound Healing and Graft Survival.

Burn injury increases the risk of morbidity and mortality by promoting severe hemodynamic shock and risk for local or systemic infection. Graft failure due to poor wound healing or infection remains a significant problem for burn subjects. The mechanisms by which local burn injury compromises the epithelial antimicrobial barrier function in the burn margin, containing the elements necessary for healing of the burn site, and in distal unburned skin, which serves as potential donor tissue, are largely unknown. The objective of this study was to establish defects in epidermal barrier function in human donor skin and burn margin, to identify potential mechanisms that may lead to graft failure and/or impaired burn wound healing. In this study, we established that epidermal lipids and respective lipid synthesis enzymes were significantly reduced in both donor skin and burn margin. We further identified diverse changes in the gene expression and protein production of several candidate skin antimicrobial peptides (AMPs) in both donor skin and burn margin. These results also parallel changes in cutaneous AMP activity against common burn wound pathogens, aberrant production of epidermal proteases known to regulate barrier permeability and AMP activity, and greater production of proinflammatory cytokines known to be induced by AMPs. These findings suggest that impaired epidermal lipid and AMP regulation could contribute to graft failure and infectious complications in subjects with burn or other traumatic injury.

Increased doxorubicin uptake and toxicity in multicellular tumour spheroids treated with DC electrical fields.

Electrochemotherapy (ECT) is a new approach to the treatment of tumours. In the present study, multicellular prostate tumour spheroids were treated with non-lethal direct current (DC) electrical fields, and uptake and toxicity of doxorubicin were investigated. An electrical field with a field strength of 500 Vm(-1) applied for a duration of 90 s resulted in neither reversible nor irreversible membrane breakdown as revealed by fluid phase uptake studies of the membrane impermeant tracer Lucifer yellow. However, treated spheroids showed an increased uptake of doxorubicin and, consequently, an increased toxicity following electrical field exposure. The electrical field raised intracellular reactive oxygen species (ROS) as revealed using 2',7'-dichlorofluorescein diacetate (H2DCFDA) as an indicator. ROS induced membrane lipid peroxidation since the lipid peroxidation end products malondialdehyde (MDA) and 4-hydroxy-2-(E)-nonenal (4-HNE) were detected after electrical field treatment. Moreover, lipid peroxidation decreased the lipid diffusion coefficient D from 4.2 x 10(-10) cm2 s(-1) to 2.7 x 10(-10) cm2 s(-1) in the control and treated sample, respectively, as revealed by fluorescence recovery after photobleaching (FRAP) experiments. The field effects could be mimicked by incubating spheroids with 100 nM hydrogen peroxide and were inhibited by the radical scavengers dehydroascorbate (DHA) and alpha-tocopherol (vitamin E), indicating that the increased uptake of doxorubicin after electrical field treatment is owing to lipid peroxidation and decreased membrane lipid mobility. Treatment of tumours with low intensity electrical fields may be useful to improve the cytotoxic capacity of anthracyclines.

Trafficking of liposomal antigen to the trans-Golgi of murine macrophages requires both liposomal lipid and liposomal protein.

Major histocompatibility complex (MHC) class I molecules found on antigen-presenting cells present peptides derived from cytoplasmic proteins to T cells. In contrast, peptides from exogenous proteins are mostly presented by class II molecules. It has been well established that liposomes can serve as an efficient delivery system for entry of exogenous protein antigens into the MHC class I pathway. Our previous studies utilizing fluorophore-labeled proteins encapsulated in liposomes demonstrated that after phagocytosis of the liposomes by bone marrow-derived macrophages (BMs), the processed peptides were subsequently visualized in the trans-Golgi, while free conalbumin was excluded from the trans-Golgi area. In the present study, we investigated whether liposomal lipids follow the same intracellular route as the liposomal proteins after phagocytosis by BMs. Multilamellar liposomes with different lipid compositions that also contained fluorescent phospholipids (empty liposomes) were incubated with murine BMs. Our results indicate that although empty liposomes were avidly phagocytosed by macrophages, the fluorescent liposomal lipids did not localize to any particular area of the cell but were distributed throughout the cell. In contrast, when a protein was encapsulated in the liposomes, the liposomal lipids were no longer dispersed throughout the cell, but were concentrated and localized in the trans-Golgi area. Furthermore, when the liposomes contained a fluorescent-labeled protein, the fluorescent peptides also localized to the trans-Golgi. These results demonstrate that the combination of both liposomal lipids and liposomal protein is required for Golgi-specific targeting of liposomal antigens. Transport of both liposomal lipids and liposomal proteins to the Golgi complex, a major subcellular organelle in the passage of MHC class I molecules, might explain why antigens encapsulated in liposomes readily induce cytotoxic T lymphocytes.