Airborne indoor allergen serine proteases and their contribution to sensitisation and activation of innate immunity in allergic airway disease.

Common airborne allergens (pollen, animal dander and those from fungi and insects) are the main triggers of type I allergic disorder in the respiratory system and are associated with allergic rhinitis, allergic asthma, as well as immunoglobulin E (IgE)-mediated allergic bronchopulmonary aspergillosis. These allergens promote IgE crosslinking, vasodilation, infiltration of inflammatory cells, mucosal barrier dysfunction, extracellular matrix deposition and smooth muscle spasm, which collectively cause remodelling of the airways. Fungus and insect (house dust mite and cockroaches) indoor allergens are particularly rich in proteases. Indeed, more than 40 different types of aeroallergen proteases, which have both IgE-neutralising and tissue-destructive activities, have been documented in the Allergen Nomenclature database. Of all the inhaled protease allergens, 85% are classed as serine protease activities and include trypsin-like, chymotrypsin-like and collagenolytic serine proteases. In this article, we review and compare the allergenicity and proteolytic effect of allergen serine proteases as listed in the Allergen Nomenclature and MEROPS databases and highlight their contribution to allergic sensitisation, disruption of the epithelial barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of allergen serine proteases as a potential treatment strategy for allergic airways disease will also be discussed.

House dust mite allergens induce Ca2+ signalling and alarmin responses in asthma airway epithelial cells.

Type 2 inflammation in asthma develops with exposure to stimuli to include inhaled allergens from house dust mites (HDM). Features include mucus hypersecretion and the formation of pro-secretory ion transport characterised by elevated basal Cl- current. Studies using human sinonasal epithelial cells treated with HDM extract report a higher protease activated receptor-2 (PAR-2) agonist-induced calcium mobilisation that may be related to airway sensitisation by allergen-associated proteases. Herein, this study aimed to investigate the effect of HDM on Ca2+ signalling and inflammatory responses in asthmatic airway epithelial cells. Primary bronchial epithelial cells (hPBECs) from asthma donors cultured at air-liquid interface were used to assess electrophysiological, Ca2+ signalling and inflammatory responses. Differences were observed regarding Ca2+ signalling in response to PAR-2 agonist 2-Furoyl-LIGRLO-amide (2-FLI), and equivalent short-circuit current (Ieq) in response to trypsin and 2-FLI, in ALI-asthma and healthy hPBECs. HDM treatment led to increased levels of intracellular cations (Ca2+, Na+) and significantly reduced the 2-FLI-induced change of Ieq in asthma cells. Apical HDM-induced Ca2+ mobilisation was found to mainly involve the activation of PAR-2 and PAR-4-associated store-operated Ca2+ influx and TRPV1. In contrast, PAR-2, PAR-4 antagonists and TRPV1 antagonist only showed slight impact on basolateral HDM-induced Ca2+ mobilisation. HDM trypsin-like serine proteases were the main components leading to non-amiloride sensitive Ieq and also increased interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP) from asthma hPBECs. These studies add further insight into the complex mechanisms associated with HDM-induced alterations in cell signalling and their relevance to pathological changes within asthma.

An audit of the use of CT pituitary scans to exclude a pituitary macroadenoma.

BACKGROUND: Pituitary imaging is often required to exclude an adenoma suspected clinically or biochemically. Although magnetic resonance (MR) is the gold standard, computerised tomography (CT) is faster, cheaper and induces less claustrophobia. Our audit at Auckland City Hospital, New Zealand, investigated whether the use of CT of the pituitary as the first line imaging to assess for a pituitary macroadenoma reduces the need for MR. METHODS: We investigated the usefulness of CT pituitary imaging in the exclusion of pituitary macroadenoma between 2012 and 2020. A re-audit was then undertaken for a period of one year between March 2021 and March 2022 to assess outcomes once a departmental policy change was implemented. At Auckland City Hospital, 32 patients across eight years were eligible for this analysis, of which 31 had data available. In our re-audit, 29 patients were eligible for this analysis. We collected data on patient demographics, relevant hormone levels, indication for imaging and imaging results and subsequent management. RESULTS: After CT pituitary imaging, 28/31 (90%) of patients did not require further imaging because the clinical question had been addressed. One year after routine initial CT pituitary imaging was implemented by the Auckland City Hospital Endocrinology Department, 29 CT pituitary scans were performed to exclude a pituitary macroadenoma. Of these patients one required further imaging due to the finding of an expanded pituitary sella but not a pituitary macroadenoma. CONCLUSION: CT pituitary imaging to exclude a pituitary macroadenoma is a useful test that may reduce the need for MR pituitary scans. TRIAL REGISTRATION: Not applicable. This was an audit as defined by the New Zealand National Ethics Advisory Committee guidelines. Please see 'Declarations' section.

The Role of Patient Engagement in Surgical Site Infection Reduction: A Process Improvement Project.

BACKGROUND: Surgical site infections (SSIs) are the second most common healthcare-associated infection, with prevention being a high-priority goal for all healthcare organizations. Although routine surveillance and standardized prevention protocols have long been used, patient engagement is an additional intervention that should be considered and may be beneficial in SSI prevention. OBJECTIVE: To determine if the development of a standardized patient education discharge plan for management of a surgical site and/or surgical drain would contribute to a reduction in SSI rates in inpatients undergoing colorectal, plastic, or general surgery. METHODS: A preintervention/postintervention design was used. Before intervention, patients and surgeons were surveyed regarding various discharge practices related to surgical incision/drain care. The intervention consisted of implementing a standardized discharge plan including standardized education and patient discharge kits. After implementation, patients were surveyed regarding discharge practices. Patient survey responses and SSI rates were compared between the preintervention and postintervention time frames. RESULTS: Rates of SSIs decreased across all three surgical specialties during the project period: colorectal SSIs decreased from 3.2% to 2.7%, plastics from 1.2% to 0.5%, and general from 0.86% to 0.33%. Improvements were also realized in patient survey responses to various aspects of surgical incision/drain care. CONCLUSIONS: Patient engagement may be an important strategy to integrate with SSI evidence-based care bundles. Active engagement of surgical patients perioperatively has the potential to improve the patient experience, which ultimately can result in improved healthcare outcomes for this population.

Furin as a therapeutic target in cystic fibrosis airways disease.

Clinical management of cystic fibrosis (CF) has been greatly improved by the development of small molecule modulators of the CF transmembrane conductance regulator (CFTR). These drugs help to address some of the basic genetic defects of CFTR; however, no suitable CFTR modulators exist for 10% of people with CF (PWCF). An alternative, mutation-agnostic therapeutic approach is therefore still required. In CF airways, elevated levels of the proprotein convertase furin contribute to the dysregulation of key processes that drive disease pathogenesis. Furin plays a critical role in the proteolytic activation of the epithelial sodium channel; hyperactivity of which causes airways dehydration and loss of effective mucociliary clearance. Furin is also responsible for the processing of transforming growth factor-ß, which is increased in bronchoalveolar lavage fluid from PWCF and is associated with neutrophilic inflammation and reduced pulmonary function. Pathogenic substrates of furin include Pseudomonas exotoxin A, a major toxic product associated with Pseudomonas aeruginosa infection and the spike glycoprotein of severe acute respiratory syndrome coronavirus 2, the causative pathogen for coronavirus disease 2019. In this review we discuss the importance of furin substrates in the progression of CF airways disease and highlight selective furin inhibition as a therapeutic strategy to provide clinical benefit to all PWCF.

A highly selective, cell-permeable furin inhibitor BOS-318 rescues key features of cystic fibrosis airway disease.

In cystic fibrosis (CF), excessive furin activity plays a critical role in the activation of the epithelial sodium channel (ENaC), dysregulation of which contributes to airway dehydration, ineffective mucociliary clearance (MCC), and mucus obstruction. Here, we report a highly selective, cell-permeable furin inhibitor, BOS-318, that derives selectivity by eliciting the formation of a new, unexpected binding pocket independent of the active site catalytic triad. Using human ex vivo models, BOS-318 showed significant suppression of ENaC, which led to enhanced airway hydration and an ∼30-fold increase in MCC rate. Furin inhibition also protected ENaC from subsequent activation by neutrophil elastase, a soluble protease dominant in CF airways. Additional therapeutic benefits include protection against epithelial cell death induced by Pseudomonas aeruginosa exotoxin A. Our findings demonstrate the utility of selective furin inhibition as a mutation-agnostic approach that can correct features of CF airway pathophysiology in a manner expected to deliver therapeutic value.

Patient Experience of Physical Restraint in the Acute Setting: A Systematic Review of the Qualitative Research Evidence.

The use of physical restraint is a contentious practice in the acute mental health setting. There are a plethora of guidelines surrounding the safe use of restraint. However, there is a scarcity of literature dedicated specifically from the perspective of the patient. The existing literature suggests that there are serious physical and psychological implications associated with the use of physical restraint for both mental health patients and nurses alike. The debate surrounding this practice has been intensified by the compelling evidence suggesting that the application of restraint is not compatible with the values of recovery in mental health care. To influence clinical practice that governs the use of restraint, it is essential to explore patient experiences of its use. Therefore, this review aims to explore patient experiences of physical restraint in the acute setting. PubMed, CINAHL and PsycINFO were systematically searched using keywords; "physical restraint" and "patient experience" and "acute setting". The search yielded n = 482 papers in total. Following the application of rigorous inclusion and exclusion criteria and data extraction, a total of n = 9 papers were considered suitable for the systematic review. Quality was assessed using the MMAT instrument. Following analysis, three themes were identified: 'the bio-psychosocial impact of restraint on patients', 'the impact of restraint on the therapeutic relationship' and 'patient needs concerning the use of restraint'. Future research opportunities have been identified that will add to the body of evidence in developing appropriate health interventions and supports for this population.

Modulation of Ion Transport to Restore Airway Hydration in Cystic Fibrosis.

Cystic fibrosis (CF) is a life-limiting genetic disorder caused by loss-of-function mutations in the gene which codes for the CF transmembrane conductance regulator (CFTR) Cl- channel. Loss of Cl- secretion across the apical membrane of airway lining epithelial cells results in dehydration of the airway surface liquid (ASL) layer which impairs mucociliary clearance (MCC), and as a consequence promotes bacterial infection and inflammation of the airways. Interventions that restore airway hydration are known to improve MCC. Here we review the ion channels present at the luminal surface of airway epithelial cells that may be targeted to improve airway hydration and MCC in CF airways.

Characterization of Clozapine-Responsive Human T Cells.

Use of the atypical antipsychotic clozapine is associated with life-threatening agranulocytosis. The delayed onset and the association with HLA variants are characteristic of an immunological mechanism. The objective of this study was to generate clozapine-specific T cell clones (TCC) and characterize pathways of T cell activation and cross-reactivity with clozapine metabolites and olanzapine. TCC were established and characterized by culturing PBMCs from healthy donors and patients with a history of clozapine-induced agranulocytosis. Modeling was used to explore the drug-HLA binding interaction. Global TCC protein changes were profiled by mass spectrometry. Six well-growing clozapine-responsive CD4+ and CD8+ TCC were used for experiments; activation of TCC required APC, with clozapine interacting directly at therapeutic concentrations with several HLA-DR molecules. TCC were also activated with N-desmethylclozapine and olanzapine at supratherapeutic concentrations. Marked changes in TCC protein expression profiles were observed when clozapine treatment was compared with olanzapine and the medium control. Docking of the compounds into the HLA-DRB1*15:01 and HLA-DRB1*04:01 binding clefts revealed that clozapine and olanzapine bind in a similar conformation to the P4-P6 peptide binding pockets, whereas clozapine N-oxide, which did not activate the TCC, bound in a different conformation. TCC secreted Th1, Th2, and Th22 cytokines and effector molecules and expressed TCR Vß 5.1, 16, 20, and 22 as well as chemokine receptors CXCR3, CCR6, CCR4, and CCR9. Collectively, these data show that clozapine interacts at therapeutic concentrations with HLA-DR molecules and activates human CD4+ T cells. Olanzapine only activates TCC at supratherapeutic concentrations.

A Novel Serine Protease Inhibitor PE-BBI Ameliorates Cockroach Extract-Mediated Airway Epithelial Barrier Dysfunction.

Epithelial barrier dysfunction, characteristic of allergic airway disease may be, at least in part, due to the action of allergen-associated protease activities. Cockroach allergy is a major global health issue, with cockroaches containing considerable serine trypsin-like protease (TLP) activity. The present study sought to evaluate two novel protease inhibitors (PE-BBI and pLR-HL), recently isolated from amphibian skin secretions, for their potential to neutralise cockroach TLP activity and to determine any protective effect on cockroach-induced airway epithelial barrier disruption. Inhibitor potencies against the cockroach-associated activities were determined using a fluorogenic peptide substrate-based activity assay. 16HBE14o- cells (16HBE; a bronchial epithelial cell line) were treated with cockroach extract (CRE) in the presence or absence of the compounds in order to assess cell viability (RealTime Glo luminescent assay) and epithelial barrier disruption (transepithelial resistance and paracellular dextran flux). PE-BBI potently and selectively inhibited CRE TLP activity (pIC50 -8), but not host (16HBE) cell surface activity, which conferred protection of 16HBE cells from CRE-induced cell damage and barrier disruption. Novel protease inhibitor strategies such as PE-BBI may be useful for the treatment of allergic airway disease caused by cockroach proteases.

Sputum trypsin-like protease activity relates to clinical outcome in cystic fibrosis.

BACKGROUND: In cystic fibrosis (CF) airways excessive levels of serine trypsin-like proteases (TLPs) activate the epithelial sodium channel (ENaC) resulting in airways dehydration and promotion of mucus secretion. Despite this the relationship of TLP activity and clinical outcome has not been studied. METHODS: We analysed supernatant (sol) prepared from CF sputum from adult CF patients in two study cohorts (29 and 33 samples, respectively). Protease activities were determined by measuring the hydrolysis of peptide-based substrates or by ELISA. Lung function was assessed by spirometry (FEV1). Mortality data was retrospectively obtained and time in months until death or transplantation used for subsequent survival analysis. RESULTS: TLP activity inversely correlated with percent predicted FEV1 (r = -0.4, p = 0.03) and was greater in individuals who did not survive beyond 5-years from the time of sample collection. A Kaplan-Meier analysis demonstrated significantly reduced survival (p = 0.04) for individuals with high TLP activity [hazard ratio (HR) of 7.21 per log unit TLP activity (p = 0.03)]. In contrast, neutrophil elastase displayed no significant associations with lung function or patient survival. Similar findings were evident in the second study cohort. CONCLUSIONS: Sputum TLP activity may represent a novel non-invasive biomarker and/or therapeutic target for CF lung disease.

Mechanistic Dichotomy in Bacterial Trichloroethene Dechlorination Revealed by Carbon and Chlorine Isotope Effects.

Tetrachloroethene (PCE) and trichloroethene (TCE) are significant groundwater contaminants. Microbial reductive dehalogenation at contaminated sites can produce nontoxic ethene but often stops at toxic cis-1,2-dichloroethene ( cis-DCE) or vinyl chloride (VC). The magnitude of carbon relative to chlorine isotope effects (as expressed by ΛC/Cl, the slope of δ13C versus δ37Cl regressions) was recently recognized to reveal different reduction mechanisms with vitamin B12 as a model reactant for reductive dehalogenase activity. Large ΛC/Cl values for cis-DCE reflected cob(I)alamin addition followed by protonation, whereas smaller ΛC/Cl values for PCE evidenced cob(I)alamin addition followed by Cl- elimination. This study addressed dehalogenation in actual microorganisms and observed identical large ΛC/Cl values for cis-DCE (ΛC/Cl = 10.0 to 17.8) that contrasted with identical smaller ΛC/Cl for TCE and PCE (ΛC/Cl = 2.3 to 3.8). For TCE, the trend of small ΛC/Cl could even be reversed when mixed cultures were precultivated on VC or DCEs and subsequently confronted with TCE (ΛC/Cl = 9.0 to 18.2). This observation provides explicit evidence that substrate adaptation must have selected for reductive dehalogenases with different mechanistic motifs. The patterns of ΛC/Cl are consistent with practically all studies published to date, while the difference in reaction mechanisms offers a potential answer to the long-standing question of why bioremediation frequently stalls at cis-DCE.

Comparison of Cervical Range-of-Motion Restriction and Craniofacial Tissue-Interface Pressure With 2 Adjustable and 2 Standard Cervical Collars.

STUDY DESIGN: Randomized controlled trial. OBJECTIVE: The aim of the study was to compare and contrast the restrictiveness and tissue-interface pressure (TIP) characteristics of 2 standard and 2 adjustable cervical collars. SUMMARY OF BACKGROUND DATA: This study compared the restrictiveness and TIP of 4 commercially available cervical collars (2 standard and 2 adjustable). Adjustable collars offer potential advantages of individualized fit for patients and decreased inventory for institutions. The overall goal was to determine whether the adjustable collars provided the same benefits of cervical range-of-motion (CROM) restriction as the standard collars without increasing TIP and risk of pressure-related complications. METHODS: A total of 48 adult volunteer subjects (24 men and 24 women) were fitted with 4 collars (Aspen, Aspen Vista, Miami J, and Miami J Advanced) in random order. Data collection included assessment of CROM restrictiveness and measurement of TIP on the mandible and occiput in upright and supine positions. The experimental, repeated measures design stratified the sample by body mass index (BMI) and sex. RESULTS: All collars restricted CROM as compared with no collar (P ≤ 0.001 each). Aspen was more restrictive than Aspen Vista and Miami J in 4 movement planes (P ≤ 0.003 each), but not significantly different from Miami J Advanced. The Miami J standard collar was associated with significantly lower peak TIPs on all sites and in all positions compared with Aspen (P ≤ 0.001), Miami J Advanced (P < 0.001), and Aspen Vista (P = 0.01 for mandible site and upright position, P < 0.001 for remaining sites and positions). Increased peak TIP correlated with high BMI across all collar types, but was significantly lower for the Miami J collar than the Aspen collar. CONCLUSION: All collars, compared with no collar, significantly restricted CROM. Although the collar-to-collar comparisons were statistically significant, the differences may have little clinical significance in the acutely injured trauma patient. The Miami J standard collar had the lowest overall TIP in both sites and positions. Ongoing effort should be devoted to staff education in proper sizing and fit, particularly for patients with high BMI.

Contribution of research networks to a clinical trial of antidepressants in people with dementia.

BACKGROUND: The Mental Health Research Network and Dementia and Neurodegenerative Diseases Research Network were established in the UK to increase research capacity and activity; the former in mental health generally, and the latter specifically in neurodegenerative disorders including dementia. Little evidence exists on the impact of these networks on research in mental health of older people. AIMS: To examine research network support to a multi-centre randomised controlled trial of antidepressants in people with depression superimposed on dementia. Method Qualitative questionnaires were completed by principal investigators, trial-funded research workers and clinical study officers (CSOs) of the research networks. RESULTS: Research network contribution was much valued by principal investigators and the nine research teams. CSOs boosted the recruitment campaign in a challenging environment and enhanced assessment processes. Some problems with consistency and staff turnover were raised. CONCLUSION: Research network input can make an appreciable difference to the process and outcome of a multi-centre clinical trial.

Association between elevated whole blood Epstein-Barr virus (EBV)-encoded RNA EBV polymerase chain reaction and reduced incidence of acute lung allograft rejection.

BACKGROUND: Accurate functional assessment of patient immunosuppression after solid-organ transplantation remains elusive. Despite therapeutic serum immunosuppressive drug levels many lung transplant recipients still develop allograft rejection. We investigated the hypothesis that detection of latent Epstein-Barr virus (EBV) in peripheral blood may be a functional marker for the net effects of administered immunosuppression. METHODS: A retrospective analysis was performed on data obtained from a prospective trial investigating the ability of a novel EBV polymerase chain reaction (PCR) panel for LMP (latent membrane protein 1), EBNA (EBV nuclear antigen) and EBER (EBV-encoded RNA) to predict future development of post-transplant lymphoproliferative disorder (PTLD). Thirty-one lung transplant patients were followed for up to 2 years after transplantation with EBV PCR panels performed on plasma and whole blood. Patients were assessed for occurrences of Grade 2 or higher acute rejection and episodes of infection. RESULTS: Patients with whole blood EBER-positive PCR had a statistically significant lower incidence (45% vs 83%) of Grade 2 or higher acute allograft rejection than patients with no positive assays (odds ratio [OR] = 0.17, 95% confidence interval [CI] 0.021 to 1.2, p = 0.048). Positive whole blood EBER PCR did not correlate with increased risk for infectious complications (OR = 1.6, 95% CI 0.22 to 11, p = 0.69). CONCLUSIONS: These results suggest that whole blood EBER EBV PCR load may represent an important functional measure of immunosuppression in solid-organ transplant patients.