Brita Pukstad

Cellular sources and inducers of cytokines present in acute wound fluid

Acute wounds contain many biological active molecules, including several cytokines and growth factors. However, the cellular sources of each molecule, as well as the stimuli inducing them, are poorly characterized. We quantified the levels of 27 cytokines, chemokines, and growth factors in acute wound fluid in a luminex‐based assay. The acute wound fluid contained particularly high levels of IL‐6 and IL‐8, as well as elevated levels of MCP‐1, IL‐1RA, PDGF, IP‐10, IFN‐γ, and TNF‐α. Surprisingly, the amounts of IL‐1β and IL‐10 were relatively low. To characterize the cellular sources of these molecules, we analyzed supernatants from monocytes, neutrophils, keratinocytes, fibroblasts, and endothelial cells stimulated with pro‐ and anti inflammatory cytokines, and different Toll‐like receptor (TLR) ligands. The different cell types showed overlapping but distinct patterns of production of signal molecules, as well as sensitivity to ligands. Among pro‐inflammatory cytokines, IL‐1β was the most potent inducer of signal molecule production. Furthermore, keratinocytes and endothelial cells were in particular responsive to the Toll‐like receptor‐3 ligand polyI:C. New interactions between cytokines and growth factors were revealed, which may have important roles in wound healing, including IL‐1β‐induced IFN‐γ and IL‐10‐induced VEGF.

Non-healing is associated with persistent stimulation of the innate immune response in chronic venous leg ulcers

BACKGROUND The molecular pathogenesis of chronic skin wounds is complex and not fully understood. Although these wounds are often characterized as being in a state of persistent inflammation, the impact and participation of the innate immune responses in sustaining this inflammation needs further investigation. OBJECTIVE We investigated the cytokine profiles, Toll-like receptor (TLR)-stimulating activities and the levels of the antibacterial peptide Lipocalin-2 (Lcn-2) in a series of healing and non-healing chronic venous leg ulcers (CVLUs) through a study time of 8 weeks. METHODS Wound fluids from healing and non-healing CVLUs were run on a Human Cytokine Antibody Array, and Lcn-2 levels measured with ELISA. HEK 293 cells transfected with TLR2 or TLR4 and their respective co-receptors, and human peripheral blood monocytes were then stimulated with the wound fluids from healing and non-healing venous leg ulcers. RESULTS Healing wounds were associated with decreasing levels of IL-1alpha, IL-1beta and MIP-1delta, whereas in non-healing wounds decreasing levels of IL-8 and MIP-1alpha were found. Accordingly, wound fluid from non-healing CVLUs contained persistent Lcn-2 levels and TLR2- and TLR4-stimulating activities, while, in healing wounds, the TLR-stimulating activities decreased over time with significantly diminished levels of Lcn-2 (p<0.005). CONCLUSIONS Innate immune responses contribute to the chronic inflammation in non-healing CVLUs through participation of Toll-like receptors. The levels of the antimicrobial peptide Lcn-2 in wound fluids from these ulcers are elevated as a reflection of this contribution.

Producing ultrapure wood cellulose nanofibrils and evaluating the cytotoxicity using human skin cells

Wood cellulose nanofibrils (CNF) have been suggested as a potential wound healing material, but its utilization is limited by FDA requirements regarding endotoxin levels. In this study a method using sodium hydroxide followed by TEMPO mediated oxidation was developed to produce ultrapure cellulose nanofibrils, with an endotoxin level of 45 endotoxin units/g (EU/g) cellulose. Scanning transmission electron microscopy (S(T)EM) revealed a highly nanofibrillated structure (lateral width of 3.7±1.3nm). Assessment of cytotoxicity and metabolic activity on Normal Human Dermal Fibroblasts and Human Epidermal Keratinocytes was done. CNF-dispersion of 50μg/ml did not affect the cells. CNF-aerogels induced a reduction of metabolic activity by the fibroblasts and keratinocytes, but no significant cell death. Cytokine profiling revealed no induction of the 27 cytokines tested upon exposure to CNF. The moisture-holding capacity of aerogels was relatively high (∼7500%), compared to a commercially available wound dressing (∼2500%), indicating that the CNF material is promising as dressing material for management of wounds with a moderate to high amount of exudate.

Hyperspectral imaging as a diagnostic tool for chronic skin ulcers

The healing process of chronic wounds is complex, and the complete pathogenesis is not known. Diagnosis is currently based on visual inspection, biopsies and collection of samples from the wound surface. This is often time consuming, expensive and to some extent subjective procedures. Hyperspectral imaging has been shown to be a promising modality for optical diagnostics. The main objective of this study was to identify a suitable technique for reproducible classification of hyperspectral data from a wound and the surrounding tissue. Two statistical classification methods have been tested and compared to the performance of a dermatologist. Hyperspectral images (400-1000 nm) were collected from patients with venous leg ulcers using a pushbroom-scanning camera (VNIR 1600, Norsk Elektro Optikk AS).Wounds were examined regularly over 4 - 6 weeks. The patients were evaluated by a dermatologist at every appointment. One patient has been selected for presentation in this paper (female, age 53 years). The oxygen saturation of the wound area was determined by wavelength ratio metrics. Spectral angle mapping (SAM) and k-means clustering were used for classification. Automatic extraction of endmember spectra was employed to minimize human interaction. A comparison of the methods shows that k-means clustering is the most stable method over time, and shows the best overlap with the dermatologist’s assessment of the wound border. The results are assumed to be affected by the data preprocessing and chosen endmember extraction algorithm. Results indicate that it is possible to develop an automated method for reliable classification of wounds based on hyperspectral data.

Alginates induce differentiation and expression of CXCR7 and CXCL12/SDF-1 in human keratinocytes—The role of calcium†

Alginates from seaweed are used in chronic wound management, though the molecular and cellular effects of various alginate dressings are not well documented. We have developed ultrapure sodium-alginates from Pseudomonas fluorescens with different content and distribution of single guluronic acid (G) residues (0-45% G), and tested their biological activities on human primary keratinocytes (KCs). The alginates inhibited KC migration and induced expression of differentiation markers. The potency of the alginates correlated with the increasing percentage of single G residues. These findings were explained by different binding and release of ionic calcium (Ca++) from the alginates which subsequently triggered differentiation. Ca-free alginates had no effect on KC migration and differentiation, but the chemokine receptor CXCR7 was upregulated. Q-PCR revealed that also CXCL12/SDF-1, one of two known CXCR7-ligands, was induced by the alginates. Both CXCR7 and CXCL12-induction was dependent on the alginate G-content, and highest upregulation was induced by an alginate with 19% single G residues. In the epidermis, CXCR7 expression was restricted to the basal layer. This study defines two biological effects of ultrapure alginates on KCs, both being dependent on the alginate structure, and being either dependent or independent of Ca.

A Novel SimpleProbe PCR Assay for Detection of Mutations in the 23S rRNA Gene Associated with Macrolide Resistance in Mycoplasma genitalium in Clinical Samples.

ABSTRACT Macrolide-resistant strains of Mycoplasma genitalium are an increasing problem throughout the world, and the implementation of a rapid and sensitive assay for mutation detection to guide treatment is needed. Macrolide-resistant strains have been shown to contain base substitutions in positions 2058 and 2059 (Escherichia coli numbering) in region V of the 23S rRNA gene. In this study, we present a SimpleProbe PCR followed by melting curve analysis to differentiate between macrolide-resistant mutants and wild types. The assay was performed on 159 Mycoplasma genitalium-positive samples, and the results were compared with DNA sequencing. We also looked at the prevalence of macrolide-resistant strains in a Norwegian population. Of 139 samples characterized successfully by sequencing, 54 (39%) were wild types and 85 (61%) were mutants, consisting of 59 (42%) A2059G, 24 (17%) A2058G, 1 (1%) A2058T, and 1 (1%) A2059C mutation. The melting curve analysis correctly differentiated between wild-type and mutant strains in all cases, but it could not identify the different mutant types. The SimpleProbe PCR proved to be a simple, rapid, and reliable method for the detection of macrolide-resistant isolates of Mycoplasma genitalium in a clinical setting.

Mechanical characteristics of nanocellulose-PEG bionanocomposite wound dressings in wet conditions

Wood nanocellulose has been proposed for wound dressing applications partly based on its capability to form translucent films with good liquid absorption capabilities. Such properties are adequate for non-healing and chronic wounds where adequate management of exudates is a requirement. In addition, the translucency will allow to follow the wound development without the necessity to remove the dressing from the wound. Understanding the mechanical properties of nanocellulose films and dressings are also most important for tailoring optimizing wound dressing structures with adequate strength, conformability, porosity and exudate management. Mechanical properties are usually assessed in standard conditions (50% relative humidity, RH), which is not relevant in a wound management situation. In this study we have assessed the mechanical properties of three nanocellulose grades varying in the degree of nanofibrillation. The effect of nanofibrillation and of polyethylene glycol (PEG) addition, on the tensile strength, elongation and elastic modulus were assessed after 24h in water and in phosphate-buffered saline (PBS). The results reveal the behavior of the nanocellulose dressings after wetting and shed light into the development of mechanical properties in environments, which are relevant from a wound management point of view.

The interaction of wood nanocellulose dressings and the wound pathogen P. aeruginosa

Chronic wounds pose an increasingly significant worldwide economic burden (over £1 billion per annum in the UK alone). With the escalation in global obesity and diabetes, chronic wounds will increasingly be a significant cause of morbidity and mortality. Cellulose nanofibrils (CNF) are highly versatile and can be tailored with specific physical properties to produce an assortment of three-dimensional structures (hydrogels, aerogels or films), for subsequent utilization as wound dressing materials. Growth curves using CNF (diameter <20nm) in suspension demonstrated an interesting dose-dependent inhibition of bacterial growth. In addition, analysis of biofilm formation (Pseudomonas aeruginosa PAO1) on nanocellulose aerogels (20g/m2) revealed significantly less biofilm biomass with decreasing aerogel porosity and surface roughness. Importantly, virulence factor production by P. aeruginosa in the presence of nanocellulose materials, quantified for the first time, was unaffected (p>0.05) over 24h. These data demonstrate the potential of nanocellulose materials in the development of novel dressings that may afford significant clinical potential.

Oligodeoxynucleotides inhibit Toll-like receptor 3 mediated cytotoxicity and CXCL8 release in keratinocytes

Abstract:  Toll‐like receptor 3 (TLR3) is an important sensor of viral infections and injury of self in keratinocytes. In this study, we stimulated primary keratinocytes with the TLR3‐ligand polyI:C. This induced a toxic effect shown by up‐regulation of the alarmin high‐mobility group protein B1 and reduced responses in a MTT‐assay. PolyI:C was a potent inducer of proinflammatory cytokines, and both these responses and the cytotoxic effects were found to be TLR3 dependent, as demonstrated by the use of siRNA for TLR3. Interestingly, co‐stimulation with oligodeoxynucleotides (ODNs) inhibited all polyI:C induced effects. This inhibition was found to be mediated by the competition of endocytic uptake of polyI:C and ODNs. We have found polyI:C induced cytotoxicity and proinflammatory responses to be dependent of TLR3 and that this may be inhibited by ODNs. With these findings, we see a promising potential for ODNs in inhibiting TLR3‐induced responses in inflammatory skin disorders.

A combined 3D and hyperspectral method for surface imaging of wounds

Information about the size and depth of a wound and how it is developing is an important prognostic tool in wound diagnostics. In this study a two-camera vision system has been developed to collect optical properties, shape and volume of chronic skin ulcers as tool for diagnostic assistance. This system combines the functionality of 2D imaging spectroscopy and 3D stereo-photogrammetry. A high resolution hyperspectral camera and a monochromatic video frame camera were mounted on the same scanning system. Stereo images were acquired to obtain information about the wound surface geometry. A Digital Surface Model (DSM) of the wound surface was reconstructed by applying stereophotogrammetric methods. The hyperspectral image was co-registered to the monochromatic frame image and the wound border was extracted by applying spectroscopic analysis (e.g. tissue oxygenation, pigmentation, classification). The resulting DSM of the undamaged surroundings of the wound was used to reconstruct the top surface above the wound and thus the wound volume. The analyses can, if desired, be limited to a certain depth of interest like the wound bed or wound border. Simultaneous analysis of the hyperspectral data and the surface model gives a promising, new, non-invasive tool for characterization of chronic wounds. Future work will concentrate on implementation of real time analysis and improvement of the accuracy of the system.