Comparing Redox and Intracellular Signalling Responses to Cold Plasma in Wound Healing and Cancer.

Cold plasma (CP) is an ionised gas containing excited molecules and ions, radicals, and free electrons, and which emits electric fields and UV radiation. CP is potently antimicrobial, and can be applied safely to biological tissue, birthing the field of plasma medicine. Reactive oxygen and nitrogen species (RONS) produced by CP affect biological processes directly or indirectly via the modification of cellular lipids, proteins, DNA, and intracellular signalling pathways. CP can be applied at lower levels for oxidative eustress to activate cell proliferation, motility, migration, and antioxidant production in normal cells, mainly potentiated by the unfolded protein response, the nuclear factor-erythroid factor 2-related factor 2 (Nrf2)-activated antioxidant response element, and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway, which also activates nuclear factor-kappa B (NFκB). At higher CP exposures, inactivation, apoptosis, and autophagy of malignant cells can occur via the degradation of the PI3K/Akt and mitogen-activated protein kinase (MAPK)-dependent and -independent activation of the master tumour suppressor p53, leading to caspase-mediated cell death. These opposing responses validate a hormesis approach to plasma medicine. Clinical applications of CP are becoming increasingly realised in wound healing, while clinical effectiveness in tumours is currently coming to light. This review will outline advances in plasma medicine and compare the main redox and intracellular signalling responses to CP in wound healing and cancer.

Therapeutic potential of antimicrobial peptides for treatment of wound infection.

Healing of cutaneous wounds is a fundamental process required to re-establish tissue integrity, repair skin barrier function, and restore skin homeostasis. Chronic wound infection, exacerbated by the growing development of resistance to conventional therapies, hinders the skin repair process and is a serious clinical problem affecting millions of people worldwide. In the past decade, the use of antimicrobial peptides (AMPs) has attracted increasing attention as a potential novel strategy for the treatment of chronic wound infections due to their unique multifaceted mechanisms of action, and AMPs have been demonstrated to function as potent host-defense molecules that can control microbial proliferation, modulate host-immune responses, and act as endogenous mediators of wound healing. To date over 3,200 AMPs have been discovered either from living organisms or through synthetic derivation, some of which have progressed to clinical trials for the treatment of burn and wound injuries. However, progress to routine clinical use has been hindered due to AMPs' susceptibility to wound and environmental factors including changes in pH, proteolysis, hydrolysis, oxidation, and photolysis. This review will discuss the latest research focused on the development and applications of AMPs for wound infections using the latest nanotechnological approaches to improve AMP delivery, and stability to present effective combinatorial treatment for clinical applications.

Liquid crystal nanoparticle platform for increased efficacy of cationic antimicrobials against biofilm infections.

Bacterial biofilm infections tolerate high concentrations of antibiotics and are insidiously challenging to treat. Liquid crystal nanoparticles (LCNPs) advance the efficacy of tobramycin in biofilm-related infections by increasing the penetration of antibiotics across the biofilm matrix. Herewith, we develop the LCNPs as a platform technology, demonstrating that the LCNPs can increase the efficacy of two antibiotic classes (i.e. aminoglycosides and colistin) in P. aeruginosa biofilm infections. In C. elegans, the LCNPs potentiated the antimicrobial effect and significantly improved the survival of the nematodes. In mice with a full-thickness excisional wound, LCNPs were non-toxic and did not impair wound repair. Compared to the unformulated antibiotic treatment, tobramycin-LCNPs reduced the chronic bacterial load by 100-fold in the wound. This was also emulated in an ex vivo P. aeruginosa porcine wound infection model. The LCNPs represent a versatile platform technology that improves the efficacy of cationic antibiotics against biofilm infections utilizing multiple administration routes.

Increased Expression of Flightless I in Cutaneous Squamous Cell Carcinoma Affects Wnt/ß-Catenin Signaling Pathway.

Cutaneous squamous cell carcinoma (cSCC) accounts for 25% of cutaneous malignancies diagnosed in Caucasian populations. Surgical removal in combination with radiation and chemotherapy are effective treatments for cSCC. Nevertheless, the aggressive metastatic forms of cSCC still have a relatively poor patient outcome. Studies have linked actin cytoskeletal dynamics and the Wnt/ß-catenin signaling pathway as important modulators of cSCC pathogenesis. Previous studies have also shown that the actin-remodeling protein Flightless (Flii) is a negative regulator of cSCC. The aim of this study was to investigate if the functional effects of Flii on cSCC involve the Wnt/ß-catenin signaling pathway. Flii knockdown was performed using siRNA in a human late stage aggressive metastatic cSCC cell line (MET-1) alongside analysis of Flii genetic murine models of 3-methylcholanthrene induced cSCC. Flii was increased in a MET-1 cSCC cell line and reducing Flii expression led to fewer PCNA positive cells and a concomitant reduction in cellular proliferation and symmetrical division. Knockdown of Flii led to decreased ß-catenin and a decrease in the expression of the downstream effector of ß-catenin signaling protein SOX9. 3-Methylcholanthrene (MCA)-induced cSCC in Flii overexpressing mice showed increased markers of cancer metastasis including talin and keratin-14 and a significant increase in SOX9 alongside a reduction in Flii associated protein (Flap-1). Taken together, this study demonstrates a role for Flii in regulating proteins involved in cSCC proliferation and tumor progression and suggests a potential role for Flii in aggressive metastatic cSCC.

Overexpression of Flii during Murine Embryonic Development Increases Symmetrical Division of Epidermal Progenitor Cells.

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/ß-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii+/-, wild type: WT, transgenic Flii overexpressing: FliiTg/Tg) using Western blot and immunohistochemistry. Flii+/- embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. FliiTg/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and ß-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.

Immunological Memory in Imiquimod-Induced Murine Model of Psoriasiform Dermatitis.

Psoriasis is a common chronic inflammatory skin condition manifested by T cell responses and characterized by preferential recurrence at previously inflamed sites upon withdrawal of treatment. The site-specific disease memory in psoriasis has been linked to CD8+CD103+ tissue-resident memory T cells (Trm) in the epidermis which were previously thought to only provide "frontline" protection against pathogens and immunosurveillance during cancer development. In this study, we correlated the presence of a subset of the Trm cells which are also CD49a+ with disease severity in human psoriatic lesions with acute and chronic disease. Using an imiquimod (IMQ)-induced murine model of psoriasiform dermatitis, we also investigated the level of CD49a+ Trm cells in acute, chronic and resolved psoriatic lesions. Investigation of clinical human samples showed that patient disease severity highly correlated with the numbers of epidermal CD49a+ Trm cells. Additionally, this subset of Trm cells was shown to persist in resolved lesions of murine psoriasiform dermatitis once clinical disease features had subsided. Importantly, these CD49a+ Trm cells showed significantly higher levels of granzyme B (GzmB) production compared to acute disease, suggesting a potential role of CD49a+ Trm cells for psoriatic re-occurrence in resolved patients. Better understanding of epidermal CD49a+ Trm cell activity is necessary for development of advanced treatment strategies for psoriasis to permit long-term, continuous disease control.

Recombinant Leucine-Rich Repeat Flightless-Interacting Protein-1 Improves Healing of Acute Wounds through Its Effects on Proliferation Inflammation and Collagen Deposition.

Wound healing is an increasing clinical problem involving substantial morbidity, mortality, and rising health care costs. Leucine-rich repeat flightless-interacting protein-1 (LRRFIP-1) regulates toll-like receptor (TLR)-mediated inflammation, suggesting a potential role in the healing of wounds. We sought to determine the role of LRRFIP-1 in wound repair and whether the exogenous addition of recombinant LRRFIP-1 (rLRRFIP-1) affected healing responses. Using a model of full-thickness incisional acute wounds in BALB/c mice, we investigated the effect of wounding on LRRFIP-1 expression. The effect of rLRRFIP-1 on cellular proliferation, inflammation, and collagen deposition was also investigated. LRRFIP-1 was upregulated in response to wounding, was found to directly associate with flightless I (Flii), and significantly increased cellular proliferation both in vitro and in vivo. rLRRFIP-1 reduced Flii expression in wounds in vivo and resulted in significantly improved healing with a concurrent dampening of TLR4-mediated inflammation and improved collagen deposition. Additionally, decreased levels of TGF-ß1 and increased levels of TGF-ß3 were observed in rLRRFIP-1-treated wounds suggesting a possible antiscarring effect of rLRRFIP-1. Further studies are required to elucidate if the mechanisms behind LRRFIP-1 action in wound repair are independent of Flii. However, these results identify rLRRFIP-1 as a possible treatment modality for improved healing of acute wounds.

Cytoskeletal Regulation of Inflammation and Its Impact on Skin Blistering Disease Epidermolysis Bullosa Acquisita.

Actin remodelling proteins regulate cytoskeletal cell responses and are important in both innate and adaptive immunity. These responses play a major role in providing a fine balance in a cascade of biological events that results in either protective acute inflammation or chronic inflammation that leads to a host of diseases including autoimmune inflammation mediated epidermolysis bullosa acquisita (EBA). This review describes the role of the actin cytoskeleton and in particular the actin remodelling protein called Flightless I (Flii) in regulating cellular inflammatory responses and its subsequent effect on the autoimmune skin blistering disease EBA. It also outlines the potential of an antibody based therapy for decreasing Flii expression in vivo to ameliorate the symptoms associated with EBA.

Understanding the outcomes of a home nursing programme for patients with epidermolysis bullosa: an Australian perspective.

Epidermolysis bullosa (EB) consists of a spectrum of genodermatoses characterised by skin fragility and various degrees of skin and mucous membrane blistering. Minimal trauma and friction can cause extensive blistering in patients with EB, resulting in a number of complications. However, wound management is the main challenge for these patients because of a high risk of infection, fluid loss and potential development of aggressive squamous cell carcinoma (SCC). Indeed, patients with EB have an increased risk for developing skin cancers compared to the general population. In 2012, a home nursing programme was established in Australia to provide assistance to families or patients with severe forms of EB. Nursing care was provided to patients with severe EB during dressing changes in their homes over a period of 2 years. Both families of patients and nurses were surveyed periodically using a developed questionnaire to assess the benefits of this home nursing and its impact on the patients, their families and the nurses. Key findings included a perceived improvement in quality of life, a better provision of support and improved family life management. These findings are the first to highlight the benefits of this national home nursing programme for EB patients within Australia and demonstrate the continued need and benefit of home nursing for patients with severe skin blistering disorders.

Flightless I over-expression impairs skin barrier development, function and recovery following skin blistering.

Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii(+/-), WT and Flii(Tg/Tg) mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in Flii(Tg)(/Tg) adult mouse skin, while Flii(Tg/Tg) keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii(Tg/Tg) mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients.

Attenuation of flightless I improves wound healing and enhances angiogenesis in a murine model of type 1 diabetes.

AIMS/HYPOTHESIS: Skin lesions and ulcerations are severe complications of diabetes that often result in leg amputations. In this study we investigated the function of the cytoskeletal protein flightless I (FLII) in diabetic wound healing. We hypothesised that overexpression of FLII would have a negative effect on diabetic wound closure and modulation of this protein using specific FLII-neutralising antibodies (FnAb) would enhance cellular proliferation, migration and angiogenesis within the diabetic wound. METHODS: Using a streptozotocin-induced model of diabetes we investigated the effect of altered FLII levels through Flii genetic knockdown, overexpression or treatment with FnAb on wound healing. Diabetic wounds were assessed using histology, immunohistochemistry and biochemical analysis. In vitro and in vivo assays of angiogenesis were used to assess the angiogenic response. RESULTS: FLII levels were elevated in the wounds of both diabetic mice and humans. Reduction in the level of FLII improved healing of murine diabetic wounds and promoted a robust pro-angiogenic response with significantly elevated von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-positive endothelial cell infiltration. Diabetic mouse wounds treated intradermally with FnAb showed improved healing and a significantly increased rate of re-epithelialisation. FnAb improved the angiogenic response through enhanced formation of capillary tubes and functional neovasculature. Reducing the level of FLII led to increased numbers of mature blood vessels, increased recruitment of smooth muscle actin-α-positive cells and improved tight junction formation. CONCLUSIONS/INTERPRETATION: Reducing the level of FLII in a wound may be a potential therapeutic approach for the treatment of diabetic foot ulcers.

Deferiprone-Gallium-Protoporphyrin Chitogel Decreases Pseudomonas aeruginosa Biofilm Infection without Impairing Wound Healing.

Pseudomonas aeruginosa is one of the most common pathogens encountered in clinical wound infections. Clinical studies have shown that P. aeruginosa infection results in a larger wound area, inhibiting healing, and a high prevalence of antimicrobial resistance. Hydroxypyridinone-derived iron chelator Deferiprone (Def) and heme analogue Gallium-Protoporphyrin (GaPP) in a chitosan-dextran hydrogel (Chitogel) have previously been demonstrated to be effective against PAO1 and clinical isolates of P. aeruginosa in vitro. Moreover, this combination of these two agents has been shown to improve sinus surgery outcomes by quickly reducing bleeding and preventing adhesions. In this study, the efficacy of Def-GaPP Chitogel was investigated in a P. aeruginosa biofilm-infected wound murine model over 6 days. Two concentrations of Def-GaPP Chitogel were investigated: Def-GaPP high dose (10 mM Def + 500 µg/mL GaPP) and Def-GaPP low dose (5 mM Def + 200 µg/mL GaPP). The high-dose Def-GaPP treatment reduced bacterial burden in vivo from day 2, without delaying wound closure. Additionally, Def-GaPP treatment decreased wound inflammation, as demonstrated by reduced neutrophil infiltration and increased anti-inflammatory M2 macrophage presence within the wound bed to drive wound healing progression. Def-GaPP Chitogel treatment shows promising potential in reducing P. aeruginosa cutaneous infection with positive effects observed in the progression of wound healing.

Deep Eutectic Solvent Eutectogels for Delivery of Broad-Spectrum Antimicrobials.

Gel-based wound dressings have gained popularity within the healthcare industry for the prevention and treatment of bacterial and fungal infections. Gels based on deep eutectic solvents (DESs), known as eutectogels, provide a promising alternative to hydrogels as they are non-volatile and highly tunable and can solubilize therapeutic agents, including those insoluble in hydrogels. A choline chloride:glycerol-cellulose eutectogel was loaded with numerous antimicrobial agents including silver nanoparticles, black phosphorus nanoflakes, and commercially available pharmaceuticals (octenidine dihydrochloride, tetracycline hydrochloride, and fluconazole). The eutectogels caused >97% growth reduction in Gram-positive methicillin-resistant Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa bacteria and the fungal species Candida albicans.

Lipid Liquid Crystal Nanoparticles: Promising Photosensitizer Carriers for the Treatment of Infected Cutaneous Wounds.

Cutaneous chronic wounds impose a silent pandemic that affects the lives of millions worldwide. The delayed healing process is usually complicated by opportunistic bacteria that infect wounds. Staphylococcus aureus is one of the most prevalent bacteria in infected cutaneous wounds, with the ability to form antibiotic-resistant biofilms. Recently, we have demonstrated the potential of gallium protoporphyrin lipid liquid crystalline nanoparticles (GaPP-LCNP) as a photosensitizer against S. aureus biofilms in vitro. Herein, we investigate the potential of GaPP-LCNP using a pre-clinical model of infected cutaneous wounds. GaPP-LCNP showed superior antibacterial activity compared to unformulated GaPP, reducing biofilm bacterial viability by 5.5 log10 compared to 2.5 log10 in an ex vivo model, and reducing bacterial viability by 1 log10 in vivo, while unformulated GaPP failed to reduce bacterial burden. Furthermore, GaPP-LCNP significantly promoted wound healing through reduction in the bacterial burden and improved early collagen deposition. These findings pave the way for future pre-clinical investigation and treatment optimizations to translate GaPP-LCNP towards clinical application.

The development of microfluidic-based western blotting: Technical advances and future perspectives.

Over the past two decades significant technical advancement in the field of western blotting has been made possible through the utilization of microfluidic technologies. In this review we provide a critical overview of these advancements, highlighting the advantages and disadvantages of each approach. Particular attention is paid to the development of now commercially available systems, including those for single cell analysis. This review also discusses more recent developments, including algorithms for automation and/or improved quantitation, the utilization of different materials/chemistries, use of projection electrophoresis, and the development of triBlots. Finally, the review includes commentary on future advances in the field based on current developments, and the potential of these systems for use as point-of-care devices in healthcare.

Flii neutralizing antibodies improve wound healing in porcine preclinical studies.

Wound healing is an important area of widely unmet medical need, with millions of procedures carried out worldwide which could potentially benefit from a product to improve the wound repair process. Our studies investigating the actin-remodeling protein Flightless I (Flii) show it to be an important regulator of wound healing. Flii-deficient mice have enhanced wound healing in comparison to Flii overexpressing mice which have impaired wound healing. For the first time, we show that a Flightless I neutralizing monoclonal antibody (FnAb) therapy is effective in a large animal model of wound repair. Porcine 5 cm incisional and 6.25 cm(2) excisional wounds were treated with FnAb at the time of wounding and for two subsequent days. The wounds were dressed in Tegaderm dressings and left to heal by secondary intention for 7 and 35 days, respectively. At the relevant end points, the wounds were excised and processed for histological analysis. Parameters of wound area, collagen deposition, and scar appearance were analyzed. The results show that treatment with FnAb accelerates reepithelialization and improves the macroscopic appearance of early scars. FnAbs have the potential to enhance wound repair and reduce scar formation.

Overexpression of the Flii gene increases dermal-epidermal blistering in an autoimmune ColVII mouse model of epidermolysis bullosa acquisita.

Epidermolysis bullosa (EB) is a severe genetic skin fragility syndrome characterized by blister formation. The molecular basis of EB is still largely unknown and wound healing in patients suffering from EB remains a major challenge to their survival. Our previous studies have identified the actin remodelling protein Flightless I (Flii) as an important mediator of wound repair. Here we identify Flii as a novel target involved in skin blistering. Flii expression was significantly elevated in 30 patients with EB, most prominently in patients with recessive dystrophic EB (RDEB) who have defects in production of type VII collagen (ColVII). Using an autoimmune ColVII murine model of EB acquisita (EBA) and an immunocompetent-ColVII-hypomorphic genetic mouse model of RDEB together with murine Flii alleles, we investigated the contribution of Flii to EB. Overexpression of Flii produced severe blistering post-induction of EBA, while decreased Flii reduced blister severity, elevated integrin expression, and improved ColVII production. Flii(+/-) blistered skin showed reduced α-SMA, TGF-ß1, and Smad 2/3 expression, suggesting that decreasing Flii may affect fibrosis. In support of this, Flii-deficient fibroblasts from EBA mice were less able to contract collagen gels in vitro; however, addition of TGF-ß1 restored collagen contraction, suggesting an interplay between Flii and TGF-ß1. Elevated Flii gene and protein expression was further observed in the blisters of ColVII hypomorphic mice, a murine model of RDEB, suggesting that reducing Flii in blistered skin could be a potential new approach for treating patients with EB.

Practical extended use of antimicrobial silver (PExUS).

BACKGROUND: Antimicrobial silver has had a role in wound antisepsis throughout history and, with the rise in acquired antibiotic resistance, silver dressings are once again commonly used. Issues with silver dressings include the important environmental consideration of nanoparticle manufacture, and the significant financial cost of these products. One solution to these problems may be to adopt an opened-but-unused model of wound care whereby dressing materials are used in piecemeal fashion and excess stored in between dressing changes. Due to a lack of literature on the topic, this project was designed with the aim of testing the antimicrobial efficacy of available silver dressings during storage after opening. METHODS: Four commonly used silver dressings were tested for antimicrobial activity using a zone of inhibition assay against clinically important pathogens. The assay was performed on opening of dressings and repeated over 3 months in storage at 4, 25 or 37°C. Analysis was performed using repeated measures ANOVA. Swab cultures were taken at each simulated dressing change to detect microbial contamination of the dressings during storage. RESULTS: There was no effect of time or storage temperature on the zone of inhibition over the 12 week test period. No swabs taken returned culture consistent with microbial contamination of stored dressings. CONCLUSION: Opened silver dressings maintain antimicrobial activity for at least 12 weeks in storage and are resistant to contamination. An opened-but-unused model for wound care is likely to improve cost-effectiveness while preserving effectiveness and safety.