Mary Cloud B. Ammons

The expanding role of NADPH oxidases in health and disease: no longer just agents of death and destruction

The NADPH oxidase was originally identified as a key component of human innate host defence. In phagocytes, this enzyme complex is activated to produce superoxide anion and other secondarily derived ROS (reactive oxygen species), which promote killing of invading micro-organisms. However, it is now well-established that NADPH oxidase and related enzymes also participate in important cellular processes not directly related to host defence, including signal transduction, cell proliferation and apoptosis. These enzymes are present in essentially every organ system in the body and contribute to a multitude of physiological events. Although essential for human health, excess NADPH-oxidase-generated ROS can promote numerous pathological conditions. Herein, we summarize our current understanding of NADPH oxidases and provide an overview of how they contribute to specific human diseases.

In vitro susceptibility of established biofilms composed of a clinical wound isolate of Pseudomonas aeruginosa treated with lactoferrin and xylitol

The medical impact of bacterial biofilms has increased with the recognition of biofilms as a major contributor to chronic wounds such as diabetic foot ulcers, venous leg ulcers and pressure ulcers. Traditional methods of treatment have proven ineffective, therefore this article presents in vitro evidence to support the use of novel antimicrobials in the treatment of Pseudomonas aeruginosa biofilm. An in vitro biofilm model with a clinical isolate of P. aeruginosa was subjected to treatment with either lactoferrin or xylitol alone or in combination. Combined lactoferrin and xylitol treatment disrupted the structure of the P. aeruginosa biofilm and resulted in a >2log reduction in viability. In situ analysis indicated that while xylitol treatment appeared to disrupt the biofilm structure, lactoferrin treatment resulted in a greater than two-fold increase in the number of permeabilised bacterial cells. The findings presented here indicated that combined treatment with lactoferrin and xylitol significantly decreases the viability of established P. aeruginosa biofilms in vitro and that the antimicrobial mechanism of this treatment includes both biofilm structural disruption and permeablisation of bacterial membranes.

Anti-Biofilm Strategies and the Need for Innovations in Wound Care

With an aging and obese population, chronic wounds such as diabetic ulcers, pressure ulcers, and venous leg ulcers are of an increasingly relevant medical concern in the developed world. Identification of bacterial biofilm contamination as a major contributor to non-healing wounds demands biofilm-targeted strategies to treat chronic wounds. While the current standard of care has proven marginally effective, there are components of standard care that should remain part of the wound treatment regime including systemic and topical antibiotics, antiseptics, and physical debridement of biofilm and devitalized tissue. Emerging anti-biofilm strategies include novel, non-invasive means of physical debridement, chemical agent strategies, and biological agent strategies. While aging and obesity will continue to be major burdens to wound care, the emergence of wounds associated with war require investigation and biotechnology development to address biofilm strategies that manage multi-drug resistant bacteria contaminating the chronic wound. The article presents some of the recent patents related to anti-biofilm strategy in wound care.

Anti-biofilm efficacy of a lactoferrin/xylitol wound hydrogel used in combination with silver wound dressings

With an epidemic increase in obesity combined with an ageing population, chronic wounds such as diabetic foot ulcers, pressure ulcers and venous leg ulcers are an increasing clinical concern. Recent studies have shown that bacterial biofilms are a major contributor to wound bioburden and interfere with the normal wound healing process; therefore, rational design of wound therapies should include analysis of anti‐biofilm characteristics. Studies using the combined treatment of bacterial biofilms with the innate immune molecule lactoferrin and the rare sugar‐alcohol xylitol have demonstrated an antimicrobial capacity against a clinical wound isolate. Studies presented here used a colony‐drip‐flow reactor biofilm model to assess the anti‐biofilm efficacy of a lactoferrin/xylitol hydrogel used in combination with commercially available silver‐based wound dressings. Log reductions in biofilm viability are compared with a commercially available wound hydrogel used in combination with the silver‐based wound dressings. For both a single species biofilm and a dual species biofilm, the lactoferrin/xylitol hydrogel in combination with the silver wound dressing Acticoat™ had a statistically significant reduction in biofilm viability relative to the commercially available wound hydrogel. This study also demonstrated a statistical interaction between the lactoferrin/xylitol hydrogel and the silver wound dressing.

Mini-review: Lactoferrin: a bioinspired, anti-biofilm therapeutic

Medically relevant biofilms have gained a significant level of interest, in part because of the epidemic rise in obesity and an aging population in the developed world. The associated comorbidities of chronic wounds such as pressure ulcers, venous leg ulcers, and diabetic foot wounds remain recalcitrant to the therapies available currently. Development of chronicity in the wound is due primarily to an inability to complete the wound healing process owing to the presence of a bioburden, specifically bacterial biofilms. New therapies are clearly needed which specifically target biofilms. Lactoferrin is a multifaceted molecule of the innate immune system found primarily in milk. While further investigation is warranted to elucidate mechanisms of action, in vitro analyses of lactoferrin and its derivatives have demonstrated that these complex molecules are structurally and functionally well suited to address the heterogeneity of bacterial biofilms. In addition, use of lactoferrin and its derivatives has proven promising in the clinic.

Quantitative NMR metabolite profiling of methicillin-resistant and methicillin-susceptible Staphylococcus aureus discriminates between biofilm and planktonic phenotypes.

Wound bioburden in the form of colonizing biofilms is a major contributor to nonhealing wounds. Staphylococcus aureus is a Gram-positive, facultative anaerobe commonly found in chronic wounds; however, much remains unknown about the basic physiology of this opportunistic pathogen, especially with regard to the biofilm phenotype. Transcriptomic and proteomic analysis of S. aureus biofilms have suggested that S. aureus biofilms exhibit an altered metabolic state relative to the planktonic phenotype. Herein, comparisons of extracellular and intracellular metabolite profiles detected by 1H NMR were conducted for methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains grown as biofilm and planktonic cultures. Principal component analysis distinguished the biofilm phenotype from the planktonic phenotype, and factor loadings analysis identified metabolites that contributed to the statistical separation of the biofilm from the planktonic phenotype, suggesting that key features distinguishing biofilm from planktonic growth include selective amino acid uptake, lipid catabolism, butanediol fermentation, and a shift in metabolism from energy production to assembly of cell-wall components and matrix deposition. These metabolite profiles provide a basis for the development of metabolite biomarkers that distinguish between biofilm and planktonic phenotypes in S. aureus and have the potential for improved diagnostic and therapeutic use in chronic wounds.

Combined treatment of Pseudomonas aeruginosa biofilm with lactoferrin and xylitol inhibits the ability of bacteria to respond to damage resulting from lactoferrin iron chelation

With an ageing and ever more obese population, chronic wounds such as diabetic ulcers, pressure ulcers and venous leg ulcers are an increasingly relevant medical concern. Identification of bacterial biofilm contamination as a major contributor to non-healing wounds demands biofilm-targeted strategies to manage chronic wounds. Pseudomonas aeruginosa has been identified as a principal biofilm-forming opportunistic pathogen in chronic wounds. The innate immune molecule lactoferrin and the rare sugar alcohol xylitol have been demonstrated to be co-operatively efficacious against P. aeruginosa biofilms in vitro. Data presented here propose a model for the molecular mechanism behind this co-operative antimicrobial effect. Lactoferrin iron chelation was identified as the primary means by which lactoferrin destabilises the bacterial membrane. By microarray analysis, 183 differentially expressed genes of ≥ 1.5-fold difference were detected. Interestingly, differentially expressed transcripts included the operon encoding components of the pyochelin biosynthesis pathway. Furthermore, siderophore detection verified that xylitol is the component of this novel synergistic treatment that inhibits the ability of the bacteria to produce siderophores under conditions of iron restriction. The findings presented here demonstrate that whilst lactoferrin treatment of P. aeruginosa biofilms results in destabilisation of the bacterial cell membrane though iron chelation, combined treatment with lactoferrin and xylitol inhibits the ability of P. aeruginosa biofilms to respond to environmental iron restriction.

Binding of Pleomorphic Adenoma Gene-like 2 to the Tumor Necrosis Factor (TNF)-α-responsive Region of the NCF2 Promoter Regulates p67phox Expression and NADPH Oxidase Activity

NCF2, the gene encoding the NADPH oxidase cytosolic component p67phox, is up-regulated by TNF-α, and we recently mapped a region in the NCF2 promoter that was required for this TNF-α-dependent response. Because this TNF-α-responsive region (TRR) lacked recognizable transcription factor binding elements, we performed studies to identify factors involved in regulating NCF2 via the TRR. Using the TRR sequence as bait in a yeast one-hybrid screen, we identified the zinc finger transcription factor Pleomorphic Adenoma Gene-Like 2 (PLAGL2) as a candidate regulator of NCF2 expression. PLAGL2-specific antibodies were generated that detected the native and SUMO1-modified forms of endogenous PLAGL2. EMSA and DNA-binding protein affinity purification analyses demonstrated specific binding of in vitro-translated as well as endogenously expressed PLAGL2 to the TRR, and chromatin immunoprecipitation assays demonstrated enhanced binding of endogenous PLAGL2 to the TRR in vivo with TNF-α treatment. Knockdown of PLAGL2 protein inhibited up-regulation of NCF2 transcript, p67phox protein expression, and subsequent superoxide production in response to TNF-α. Furthermore, relative levels of native and SUMO1-modified endogenous PLAGL2 protein were modulated in a time-dependant manner in response to TNF-α treatment. These data clearly identify PLAGL2 as a novel regulator of NCF2 gene expression as well as NADPH oxidase activity and contribute to a greater understanding of the transcriptional regulation of NCF2.

Binding of pleomorphic adenoma gene-like 2 to the TNF-α-responsive region of the NCF2 promoter regulates p67phox expression and NADPH oxidase activity

NCF2, the gene encoding the NADPH oxidase cytosolic component p67phox, is up-regulated by TNF-α, and we recently mapped a region in the NCF2 promoter that was required for this TNF-α-dependent response. Because this TNF-α-responsive region (TRR) lacked recognizable transcription factor binding elements, we performed studies to identify factors involved in regulating NCF2 via the TRR. Using the TRR sequence as bait in a yeast one-hybrid screen, we identified the zinc finger transcription factor Pleomorphic Adenoma Gene-Like 2 (PLAGL2) as a candidate regulator of NCF2 expression. PLAGL2-specific antibodies were generated that detected the native and SUMO1-modified forms of endogenous PLAGL2. EMSA and DNA-binding protein affinity purification analyses demonstrated specific binding of in vitro-translated as well as endogenously expressed PLAGL2 to the TRR, and chromatin immunoprecipitation assays demonstrated enhanced binding of endogenous PLAGL2 to the TRR in vivo with TNF-α treatment. Knockdown of PLAGL2 protein inhibited up-regulation of NCF2 transcript, p67phox protein expression, and subsequent superoxide production in response to TNF-α. Furthermore, relative levels of native and SUMO1-modified endogenous PLAGL2 protein were modulated in a time-dependant manner in response to TNF-α treatment. These data clearly identify PLAGL2 as a novel regulator of NCF2 gene expression as well as NADPH oxidase activity and contribute to a greater understanding of the transcriptional regulation of NCF2.

Biochemical association of metabolic profile and microbiome in chronic pressure ulcer wounds.

Chronic, non-healing wounds contribute significantly to the suffering of patients with co-morbidities in the clinical population with mild to severely compromised immune systems. Normal wound healing proceeds through a well-described process. However, in chronic wounds this process seems to become dysregulated at the transition between resolution of inflammation and re-epithelialization. Bioburden in the form of colonizing bacteria is a major contributor to the delayed headlining in chronic wounds such as pressure ulcers. However how the microbiome influences the wound metabolic landscape is unknown. Here, we have used a Systems Biology approach to determine the biochemical associations between the taxonomic and metabolomic profiles of wounds colonized by bacteria. Pressure ulcer biopsies were harvested from primary chronic wounds and bisected into top and bottom sections prior to analysis of microbiome by pyrosequencing and analysis of metabolome using 1H nuclear magnetic resonance (NMR) spectroscopy. Bacterial taxonomy revealed that wounds were colonized predominantly by three main phyla, but differed significantly at the genus level. While taxonomic profiles demonstrated significant variability between wounds, metabolic profiles shared significant similarity based on the depth of the wound biopsy. Biochemical association between taxonomy and metabolic landscape indicated significant wound-to-wound similarity in metabolite enrichment sets and metabolic pathway impacts, especially with regard to amino acid metabolism. To our knowledge, this is the first demonstration of a statistically robust correlation between bacterial colonization and metabolic landscape within the chronic wound environment.