Heather M. Powell

Written action plans for asthma: an evidence-based review of the key components

Background: Written action plans for asthma facilitate the early detection and treatment of an asthma exacerbation. Several versions of action plans have been published but the key components have not been determined. A study was undertaken to determine the impact of individual components of written action plans on asthma health outcomes. Methods: Randomised controlled trials (n = 26) that evaluated asthma action plans as part of asthma self-management education were identified. Action plans were classified as being individualised and complete if they specified when and how to increase treatment (n = 17), and as incomplete (n = 4) or non-specific (n = 5) if they did not include these instructions. Results: For individualised complete written action plans the use of 2–4 action points and the use of both inhaled (ICS) and oral (OCS) corticosteroid consistently improved asthma outcomes. Action points based on personal best peak expiratory flow (PEF) consistently improved health outcomes while those based on percentage predicted PEF did not. The efficacy of incomplete action plans was inconclusive because of insufficient data. Non-specific action plans led to improvements in knowledge and symptoms. Conclusion: Individualised written action plans based on personal best PEF, using 2–4 action points, and recommending both ICS and OCS for treatment of exacerbations consistently improve asthma health outcomes. Other variations appear less beneficial or require further study. These observations provide a guide to the types of variations possible with written action plans, and strongly support the use of individualised complete written action plans.

Engineered Human Skin Fabricated Using Electrospun Collagen–PCL Blends: Morphogenesis and Mechanical Properties

Engineered human skin is commonly fabricated using collagen scaffolds that often have poor mechanical properties. To improve the strength of collagen-based scaffolds, poly(caprolactone) (PCL) was blended with collagen and formed into submicron fibers using electrospinning. At concentrations < 10% PCL (M(PCL)/[M(Collagen) + M(PCL)] x 100), the PCL component was evenly distributed within the collagen matrix. Increasing the PCL component to 30% caused separation of the collagen and PCL phases forming local domains of PCL within the collagen matrix. Tensile testing indicated that 10-100% PCL concentrations significantly improved the strength and stiffness of the acellular scaffolds. Engineered skin (ES) made with blended collagen-PCL at a concentration of up to 10% PCL did not significantly alter the stratification of the cells, cell proliferation, or epidermal differentiation compared to the 100% collagen group. Ultimate tensile strength of ES fabricated with the collagen-PCL blends was not significantly greater than that of ES made with 100% collagen scaffolds (0% PCL). The 30% PCL group had the least amount of mechanical strength likely caused by poor epidermal formation and reduced cell viability. These results indicate that minimal additions of PCL can be blended with collagen to produce scaffolds suitable for tissue engineering of human skin. However, the increase in scaffold strength with higher PCL concentrations did not result in significantly stronger ES, indicating that high cell viability and proper development of the epidermis are important factors for developing ES with high strength.

Mixed-Species Biofilm Compromises Wound Healing by Disrupting Epidermal Barrier Function

In chronic wounds, biofilm infects host tissue for extended periods of time. This work establishes the first chronic preclinical model of wound biofilm infection aimed at addressing the long‐term host response. Although biofilm‐infected wounds did not show marked differences in wound closure, the repaired skin demonstrated compromised barrier function. This observation is clinically significant, because it leads to the notion that even if a biofilm infected wound is closed, as observed visually, it may be complicated by the presence of failed skin, which is likely to be infected and/or further complicated postclosure. Study of the underlying mechanisms recognized for the first time biofilm‐inducible miR‐146a and miR‐106b in the host skin wound‐edge tissue. These miRs silenced ZO‐1 and ZO‐2 to compromise tight junction function, resulting in leaky skin as measured by transepidermal water loss (TEWL). Intervention strategies aimed at inhibiting biofilm‐inducible miRNAs may be productive in restoring the barrier function of host skin. Copyright

Chondroitin-6-Sulfate Incorporation and Mechanical Stimulation Increase MSC-Collagen Sponge Construct Stiffness

Using functional tissue engineering principles, our laboratory has produced tendon repair tissue which matches the normal patellar tendon force‐displacement curve up to 32% of failure. This repair tissue will need to withstand more strenuous activities, which can reach or even exceed 40% of failure force. To improve the linear stiffness of our tissue engineered constructs (TECs) and tissue engineered repairs, our lab is incorporating the glycosaminoglycan chondroitin‐6‐sulfate (C6S) into a type I collagen scaffold. In this study, we examined the effect of C6S incorporation and mechanical stimulation cycle number on linear stiffness and mRNA expression (collagen types I and III, decorin and fibronectin) for mesenchymal stem cell (MSC)‐collagen sponge TECs. The TECs were fabricated by inoculating MSCs at a density of 0.14 × 106 cells/construct onto pre‐cut scaffolds. Primarily type I collagen scaffold materials, with or without C6S, were cultured using mechanical stimulation with three different cycle numbers (0, 100, or 3,000 cycles/day). After 2 weeks in culture, TECs were evaluated for linear stiffness and mRNA expression. C6S incorporation and cycle number each played an important role in gene expression, but only the interaction of C6S incorporation and cycle number produced a benefit for TEC linear stiffness.

Initial starting dose of inhaled corticosteroids in adults with asthma: a systematic review

Background: Asthma guidelines vary in their recommendations for the initial dose of inhaled corticosteroid (ICS) in asthma. A systematic review of the literature was conducted to establish the optimal starting dose of ICS for asthma in adults. Methods: Randomised controlled trials comparing two doses of the same ICS in adults with asthma and no concomitant inhaled or oral corticosteroid were assessed. Included trials were analysed according to the following ICS dose comparisons: high (⩾800 μg/day beclomethasone (BDP)) versus moderate (400<800 μg/day BDP) (n = 7); moderate versus low (<400 μg/day BDP) (n = 6); step down versus constant dose (n = 4). Results: Fourteen publications describing 13 trials were included in the review. Studies (n = 4) that compared a step down approach with a constant moderate/low dose of ICS found no difference in lung function, symptoms, or rescue medications between the two treatment approaches (p>0.05). There was no difference in the change in morning peak flow after treatment with high compared with moderate dose ICS. When compared with low dose ICS, moderate dose ICS significantly improved morning peak flow (change from baseline WMD 11.14 l/min, 95% CI 1.34 to 20.93) and nocturnal symptoms (SMD −0.29, 95% CI −0.53 to −0.06). Conclusions: For patients with asthma who require ICS, starting with a moderate dose is equivalent to starting with a high dose and stepping down. The small non-significant benefits of starting with a high ICS dose are not of sufficient clinical benefit to warrant its use. Initial moderate ICS doses appear to be more effective than an initial low ICS dose.

The Effect of Azithromycin in Adults with Stable Neutrophilic COPD: A Double Blind Randomised, Placebo Controlled Trial

Background Chronic Obstructive Pulmonary Disease (COPD) is a progressive airway disease characterised by neutrophilic airway inflammation or bronchitis. Neutrophilic bronchitis is associated with both bacterial colonisation and lung function decline and is common in exacerbations of COPD. Despite current available therapies to control inflammation, neutrophilic bronchitis remains common. This study tested the hypothesis that azithromycin treatment, as an add-on to standard medication, would significantly reduce airway neutrophil and neutrophils chemokine (CXCL8) levels, as well as bacterial load. We conducted a randomised, double-blind, placebo-controlled study in COPD participants with stable neutrophilic bronchitis. Methods Eligible participants (n = 30) were randomised to azithromycin 250 mg daily or placebo for 12 weeks in addition to their standard respiratory medications. Sputum was induced at screening, randomisation and monthly for a 12 week treatment period and processed for differential cell counts, CXCL8 and neutrophil elastase assessment. Quantitative bacteriology was assessed in sputum samples at randomisation and the end of treatment visit. Severe exacerbations where symptoms increased requiring unscheduled treatment were recorded during the 12 week treatment period and for 14 weeks following treatment. A sub-group of participants underwent chest computed tomography scans (n = 15). Results Nine participants with neutrophilic bronchitis had a potentially pathogenic bacteria isolated and the median total bacterial load of all participants was 5.22×107 cfu/mL. Azithromycin treatment resulted in a non-significant reduction in sputum neutrophil proportion, CXCL8 levels and bacterial load. The mean severe exacerbation rate was 0.33 per person per 26 weeks in the azithromycin group compared to 0.93 exacerbations per person in the placebo group (incidence rate ratio (95%CI): 0.37 (0.11,1.21), p = 0.062). For participants who underwent chest CT scans, no alterations were observed. Conclusions In stable COPD with neutrophilic bronchitis, add-on azithromycin therapy showed a trend to reduced severe exacerbations sputum neutrophils, CXCL8 levels and bacterial load. Future studies with a larger sample size are warranted. Trial Registration Australian New Zealand Clinical Trials Registry ACTRN12609000259246

Regulation of electrospun scaffold stiffness via coaxial core diameter.

Scaffold mechanics influence cellular behavior, including migration, phenotype and viability. Scaffold stiffness is commonly modulated through cross-linking, polymer density, or bioactive coatings on stiff substrates. These approaches provide useful information about cellular response to substrate stiffness; however, they are not ideal as the processing can change substrate morphology, density or chemistry. Coaxial electrospinning was investigated as a fabrication method to produce scaffolds with tunable stiffness and strength without changing architecture or surface chemistry. Core solution concentration, solvent and feed rate were utilized to control core diameter with higher solution concentration and feed rate positively correlating with increased fiber diameter and stiffness. Coaxial scaffolds electrospun with an 8 wt./vol.% polycaprolactone (PCL)-HFP solution at 1 ml h(-1) formed scaffolds with an average core diameter of 1.1±0.2 μm and stiffness of 0.027±3.3×10(-3) N mm(-1). In contrast, fibers which were 2.6±0.1 μm in core diameter yielded scaffolds with a stiffness of 0.065±4.7×10(-3) N mm(-1). Strength and stiffness positively correlated with core diameter with no significant difference in total fiber diameter and interfiber distance observed in as-spun scaffolds. These data indicate that coaxial core diameter can be utilized to tailor mechanical properties of three-dimensional scaffolds and would provide an ideal scaffold for assessing the effect of scaffold mechanics on cell behavior.

Loss of Myoferlin Redirects Breast Cancer Cell Motility towards Collective Migration

Cell migration plays a central role in the invasion and metastasis of tumors. As cells leave the primary tumor, they undergo an epithelial to mesenchymal transition (EMT) and migrate as single cells. Epithelial tumor cells may also migrate in a highly directional manner as a collective group in some settings. We previously discovered that myoferlin (MYOF) is overexpressed in breast cancer cells and depletion of MYOF results in a mesenchymal to epithelial transition (MET) and reduced invasion through extracellular matrix (ECM). However, the biomechanical mechanisms governing cell motility during MYOF depletion are poorly understood. We first demonstrated that lentivirus-driven shRNA-induced MYOF loss in MDA-MB-231 breast cancer cells (MDA-231MYOF-KD) leads to an epithelial morphology compared to the mesenchymal morphology observed in control (MDA- 231LTVC) and wild-type cells. Knockdown of MYOF led to significant reductions in cell migration velocity and MDA- 231MYOF-KD cells migrated directionally and collectively, while MDA-231LTVC cells exhibited single cell migration. Decreased migration velocity and collective migration were accompanied by significant changes in cell mechanics. MDA-231MYOF-KD cells exhibited a 2-fold decrease in cell stiffness, a 2-fold increase in cell-substrate adhesion and a 1.5-fold decrease in traction force generation. In vivo studies demonstrated that when immunocompromised mice were implanted with MDA- 231MYOF-KD cells, tumors were smaller and demonstrated lower tumor burden. Moreover, MDA- 231MYOF-KD tumors were highly circularized and did not invade locally into the adventia in contrast to MDA- 231LTVC-injected animals. Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors. These data indicate that MYOF, a previously unrecognized protein in cancer, is involved in MDA-MB-231 cell migration and contributes to biomechanical alterations. Our results indicate that changes in biomechanical properties following loss of this protein may be an effective way to alter the invasive capacity of cancer cells.

Fluorescein Diacetate for Determination of Cell Viability in Tissue-Engineered Skin

Assurance of the quality of cultured skin substitutes (CSSs) currently relies on representative histology and determination of surface hydration, which provide limited sampling at selected points. To evaluate uniformity of cell density on the collagen matrices before clinical use, a field assessment of cell viability is advantageous. This study aimed to develop a field measure of cell viability in CSSs in vitro using fluorescein diacetate (FdA). CSSs were stained 3 days after keratinocyte inoculation using 0.04 mg/mL FdA followed by exposure to 366 nm of ultraviolet light. CSS fluorescence quantified using Metamorph image analysis was correlated with inoculation density, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) values and histology of corresponding biopsies. CSS fluorescence correlated significantly with inoculation density (p < 0.001) and MTT values (p < 0.001) of biopsies collected immediately after FdA staining. Fluorescence at day 3 also predicted day 10 MTT values. No toxicity was detected in CSSs, and normal in vitro and in vivo histology was demonstrated after FdA exposure. In conclusion, measurement of intracellular fluorescence with FdA allows for the early, comprehensive measurement of cellular distributions and viability in engineered skin and may therefore facilitate quality assurance.

Plasma surface modification of electrospun fibers for adhesion-based cancer cell sorting

Personalized cancer therapies drive the need for devices that rapidly and accurately segregate cancer cells from solid tumors. One potential sorting strategy is to segregate populations of cells based on their relative strength of adhesion. To investigate the effect of surface hydrophilicity and cell phenotype on adhesion, primary human breast skin fibroblasts and keratinocytes and MCF-7 breast cancer cells were seeded onto air and CF(4) plasma-treated nanofibers followed by exposure to three shear stresses (200, 275 and 350 dynes per cm(2)) 1 hour after inoculation. No difference in strength of adhesion was measured in either fibroblasts or keratinocytes on either plasma treated-surface: all exhibited >60% of the initial cell count after a 5 minute exposure to 350 dynes per cm(2) of shear stress. In contrast, a significant difference between relative strength of adhesion on air versus CF(4) plasma-treated surfaces was observed for MCF-7 cells: 26% and 6.6% of cells remained on the air and CF(4) plasma-treated surfaces, respectively. The ability to sort this cancer cell line from two non-cancerous primary human cells was evaluated by inoculating a mixture of all three cell types simultaneously onto CF(4) treated nanofibers followed by 1 hour of culture and exposure to 350 dynes per cm(2) shear stress. The majority of MCF-7 cells were removed (0.7% remained) while a majority of fibroblasts and keratinocytes remained adhered (74 and 57%). Post-sorted MCF-7 viability and morphology remained unchanged, preserving the possibility of post-separation and analysis. These data suggest that the plasma treatment of electrospun scaffolds provides a tool useful in sorting cancer cells from a mixed cell population based on adhesion strength.