Baculovirus entry into the central nervous system of Spodoptera exigua caterpillars is independent of the viral protein tyrosine phosphatase.

Neuroparasitism concerns the hostile take-over of a host's nervous system by a foreign invader, in order to alter the behaviour of the host in favour of the parasite. One of the most remarkable cases of parasite-induced host behavioural manipulation comprises the changes baculoviruses induce in their caterpillar hosts. Baculoviruses may manipulate caterpillar behaviour in two ways: hyperactivity (increased movement in the horizontal plane) and/or tree-top disease (movement to elevated levels in the vertical plane). Those behavioural changes are followed by liquefaction and death of the caterpillar. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Spodoptera exigua caterpillars, an enzymatic active form of the virally encoded protein tyrosine phosphatase (PTP) is needed for the expression of hyperactivity from 3 days post infection (dpi). Using eGFP-expressing recombinant AcMNPV strains, we show that infection of the caterpillar's central nervous system (CNS) can be observed primarily from 3 dpi onwards. In addition, we demonstrate that the structural and enzymatic function of PTP does not play a role in infection of the CNS. Instead we show that the virus entered the CNS via the trachea, progressing caudally to frontally through the CNS and that the infection progressed from the outermost cell layers towards the inner cell layers of the CNS, in a PTP independent manner. These findings help to further understand parasitic manipulation and the mechanisms by which neuroparasites infect the host nervous system to manipulate host behaviour.

Identifying critical inhalation technique errors in Dry Powder Inhaler use in patients with COPD based on the association with health status and exacerbations: findings from the multi-country cross-sectional observational PIFotal study.

BACKGROUND: Correct inhaler use depends on a complex interplay of factors, including device preparation and generating sufficient inspiratory flow. It is currently unknown which inhalation technique errors can be considered critical in Chronic Obstructive Pulmonary Disease (COPD) patients on Dry Powder Inhaler (DPI) maintenance therapy. OBJECTIVE: To investigate the association between inhalation technique errors and health status or exacerbations in patients with COPD. Additionally, the association between the number of errors and COPD outcomes was determined. METHODS: The PIFotal study is a cross-sectional multi-country observational study in a primary care setting, including 1434 COPD patients aged ≥ 40 years (50.1% female; mean age 69.2 yrs) using a DPI for their maintenance therapy. Inhalation technique was video recorded and scored by two independent researchers using inhaler-specific checklists. Health status was assessed with two questionnaires; the Clinical COPD Questionnaire (CCQ) and the COPD Assessment Test (CAT). The number of moderate and severe exacerbations in the past 12 months was recorded. Critical errors were identified based on their association with health status or exacerbations through multi-level prediction models adjusted for identified confounding. RESULTS: Errors in inhalation technique steps 'Breathe in', 'Hold breath', and 'Breathe out calmly after inhalation' were significantly associated with poorer CCQ and CAT outcomes and thus deemed critical. None of the errors were significantly associated with moderate exacerbations. Patients with errors 'Preparation', 'Hold inhaler in correct position during inhalation', and 'Breathe in' had significantly more severe exacerbations, and therefore these errors were also deemed critical. 81.3% of patients with COPD made at least one critical error. Specific combinations of errors were associated with worse outcomes. The more inhalation technique errors identified, the poorer the health status and the higher the exacerbation rate. CONCLUSION: In this study, we identified multiple critical inhalation technique errors in COPD patients using DPIs each associated with poorer outcomes. Explorative analysis revealed that specific combinations of errors may be of clinical relevance, especially those related to the inhalation manoeuvre. COPD outcomes worsened with increasing error count. These results warrant further prospective longitudinal studies to establish the effect of correcting these errors on COPD control. TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT04532853 (31/08/2020).

Clinical recommendations for dry powder inhaler use in the management of COPD in primary care.

Over 1400 patients using dry powder inhalers (DPIs) to deliver COPD maintenance therapies were recruited across Europe and Australia. Their peak inspiratory flow (PIF) was measured, inhaler technique was observed, and adherence to treatment assessed. From relating the findings with patient health status, and thereby identifying critical errors, key clinical recommendations for primary care clinicians were determined, namely - measure PIF before prescribing a DPI to ensure inhalation manoeuvre ability is well-matched with the device. Some patients could benefit from inhalation training whereas others should have their DPI changed for one better suited to their inspiratory ability or alternatively be prescribed an active device (such as a soft mist inhaler or pressurized metered dose inhaler). Observing the inhalation technique was valuable however this misses suboptimal PIF (approaching one fourth of patients with a satisfactory observed manoeuvre had a suboptimal PIF for their DPI). Assess adherence as deliberate non-adherence can point to a mismatch between a patient and their inhaler (deliberate non-adherence was significantly associated with PIFs below the minimum for the DPI). In-person observation of inhalation technique was found to be inferior to video rating based on device-specific checklists. Where video assessments are not possible, observation training for healthcare professionals would therefore be valuable particularly to improve the ability to identify the critical errors associated with health status namely 'teeth and lips sealed around mouthpiece', 'breathe in' and 'breathing out calmly after inhalation'. However, it is recommended that observation alone should not replace PIF measurement in the DPI selection process.Trial registration: https://clinicaltrials.gov/ct2/show/NCT04532853 .

Suboptimal Peak Inspiratory Flow and Critical Inhalation Errors are Associated with Higher COPD-Related Healthcare Costs.

Purpose: To assess the relationship between suboptimal Peak Inspiratory Flow (sPIF), inhalation technique errors, and non-adherence, with Healthcare Resource Utilisation (HCRU) in Chronic Obstructive Pulmonary Disease (COPD) patients receiving maintenance therapy via a Dry Powder Inhaler (DPI). Patients and methods: The cross-sectional, multi-country PIFotal study included 1434 COPD patients (≥40 years) using a DPI for maintenance therapy. PIF was measured with the In-Check DIAL G16, and sPIF was defined as a typical PIF lower than required for the device. Inhalation technique was assessed by standardised evaluation of video recordings and grouped into 10 steps. Patients completed the "Test of Adherence to Inhalers" questionnaire. HCRU was operationalised as COPD-related costs for primary healthcare, secondary healthcare, medication, and total COPD-related costs in a 1-year period. Results: Participants with sPIF had higher medication costs compared with those with optimal PIF (cost ratio [CR]: 1.07, 95% CI [1.01, 1.14]). Multiple inhalation technique errors were associated with increased HCRU. Specifically, "insufficient inspiratory effort" with higher secondary healthcare costs (CR: 2.20, 95% CI [1.37, 3.54]) and higher total COPD-related costs (CR: 1.16, 95% CI 1.03-1.31). "no breath-hold following the inhalation manoeuvre (<6 s)" with higher medication costs (CR: 1.08, 95% CI [1.02, 1.15]) and total COPD-related costs (CR 1.17, 95% CI [1.07, 1.28]), and "not breathing out calmly after inhalation" with higher medication costs (CR: 1.19, 95% CI [1.04, 1.37]). Non-adherence was not significantly associated with HCRU. Conclusion: sPIF and inhalation technique errors were associated with higher COPD-related healthcare utilisation and costs in COPD patients on DPI maintenance therapy.

Factors associated with health status and exacerbations in COPD maintenance therapy with dry powder inhalers.

The study aimed to determine the associations of Peak Inspiratory Flow (PIF), inhalation technique and adherence with health status and exacerbations in participants with COPD using DPI maintenance therapy. This cross-sectional multi-country observational real-world study included COPD participants aged ≥40 years using a DPI for maintenance therapy. PIF was measured three times with the In-Check DIAL G16: (1) typical PIF at resistance of participant's inhaler, (2) maximal PIF at resistance of participant's inhaler, (3) maximal PIF at low resistance. Suboptimal PIF (sPIF) was defined as PIF lower than required for the device. Participants completed questionnaires on health status (Clinical COPD Questionnaire (CCQ)), adherence (Test of Adherence to Inhalers (TAI)) and exacerbations. Inhalation technique was assessed by standardised evaluation of video recordings. Complete data were available from 1434 participants (50.1% female, mean age 69.2 years). GOLD stage was available for 801 participants: GOLD stage I (23.6%), II (54.9%), III (17.4%) and IV (4.1%)). Of all participants, 29% had a sPIF, and 16% were shown able to generate an optimal PIF but failed to do so. sPIF was significantly associated with worse health status (0.226 (95% CI 0.107-0.346), worse units on CCQ; p = 0.001). The errors 'teeth and lips sealed around mouthpiece', 'breathe in', and 'breathe out calmly after inhalation' were related to health status. Adherence was not associated with health status. After correcting for multiple testing, no significant association was found with moderate or severe exacerbations in the last 12 months. To conclude, sPIF is associated with poorer health status. This study demonstrates the importance of PIF assessment in DPI inhalation therapy. Healthcare professionals should consider selecting appropriate inhalers in cases of sPIF.

Topography-Mediated Myotube and Endothelial Alignment, Differentiation, and Extracellular Matrix Organization for Skeletal Muscle Engineering.

Understanding the response of endothelial cells to aligned myotubes is important to create an appropriate environment for tissue-engineered vascularized skeletal muscle. Part of the native tissue environment is the extracellular matrix (ECM). The ECM is a supportive scaffold for cells and allows cellular processes such as proliferation, differentiation, and migration. Interstitial matrix and basal membrane both comprise proteinaceous and polysaccharide components for strength, architecture, and volume retention. Virtually all cells are anchored to their basal lamina. One of the physical factors that affects cell behavior is topography, which plays an important role on cell alignment. We tested the hypothesis that topography-driven aligned human myotubes promote and support vascular network formation as a prelude to in vitro engineered vascularized skeletal muscle. Therefore, we used a PDMS-based topography substrate to investigate the influence of pre-aligned myotubes on the network formation of microvascular endothelial cells. The aligned myotubes produced a network of collagen fibers and laminin. This network supported early stages of endothelial network formation.

Topography-driven alterations in endothelial cell phenotype and contact guidance.

Understanding how endothelial cell phenotype is affected by topography could improve the design of new tools for tissue engineering as many tissue engineering approaches make use of topography-mediated cell stimulation. Therefore, we cultured human pulmonary microvascular endothelial cells (ECs) on a directional topographical gradient to screen the EC vascular-like network formation and alignment response to nano to microsized topographies. The cell response was evaluated by microscopy. We found that ECs formed unstable vascular-like networks that aggregated in the smaller topographies and flat parts whereas ECs themselves aligned on the larger topographies. Subsequently, we designed a mixed topography where we could explore the network formation and proliferative properties of these ECs by live imaging for three days. Vascular-like network formation continued to be unstable on the topography and were only produced on the flat areas and a fibronectin coating did not improve the network stability. However, an instructive adipose tissue-derived stromal cell (ASC) coating provided the correct environment to sustain the vascular-like networks, which were still affected by the topography underneath. It was concluded that large microsized topographies inhibit vascular endothelial network formation but not proliferation and flat and nano/microsized topographies allow formation of early networks that can be stabilized by using an ASC instructive layer.