A novel in vitro cell model of the proteinase/antiproteinase balance observed in alpha-1 antitrypsin deficiency.

Background: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting from mutations in the alpha-1 antitrypsin (AAT) protein, a major systemic antiproteinase, resulting in reduced/no release of AAT, disrupting the proteinase/antiproteinase balance. A sustained imbalance can cause structural changes to the lung parenchyma, leading to emphysema. Predicting and assessing human responses to potential therapeutic candidates from preclinical animal studies have been challenging. Our aims were to develop a more physiologically relevant in vitro model of the proteinase/antiproteinase balance and assess whether the data generated could better predict the efficacy of pharmacological candidates to inform decisions on clinical trials, together with expected biomarker responses. Methods: We developed an in vitro model assessing the proteinase/antiproteinase balance by the changes in the fibrinogen cleavage products of neutrophil elastase (NE) and proteinase 3 (PR3). This allowed the assessment of physiological and pharmaceutical neutrophil serine proteinase (NSP) inhibitors to determine the putative threshold at which the maximal effect is achieved. Results: AAT significantly reduced NE and PR3 activity footprints, with the maximal reduction achieved at concentrations above 10 µM. The inhibitor MPH966 alone also significantly reduced NE footprint generation in a concentration-dependent manner, leveling out above 100 nM but had no effect on the PR3 footprint. At levels of AAT consistent with AATD, MPH966 had an additive effect, reducing the NE activity footprint more than either inhibitor alone. Conclusion: Our results support an inhibitor threshold above which the activity footprint generation appears resistant to increasing dosage. Our model can support the testing of inhibitors, confirming activity biomarkers as indicators of likely pharmaceutical efficacy, the assessment of NSP activity in the pathophysiology of emphysema, and the likely function of biological or pharmacological inhibitors in disease management.

Neutrophils in asthma: the good, the bad and the bacteria.

Airway inflammation plays a key role in asthma pathogenesis but is heterogeneous in nature. There has been significant scientific discovery with regard to type 2-driven, eosinophil-dominated asthma, with effective therapies ranging from inhaled corticosteroids to novel biologics. However, studies suggest that approximately 1 in 5 adults with asthma have an increased proportion of neutrophils in their airways. These patients tend to be older, have potentially pathogenic airway bacteria and do not respond well to classical therapies. Currently, there are no specific therapeutic options for these patients, such as neutrophil-targeting biologics.Neutrophils comprise 70% of the total circulatory white cells and play a critical defence role during inflammatory and infective challenges. This makes them a problematic target for therapeutics. Furthermore, neutrophil functions change with age, with reduced microbial killing, increased reactive oxygen species release and reduced production of extracellular traps with advancing age. Therefore, different therapeutic strategies may be required for different age groups of patients.The pathogenesis of neutrophil-dominated airway inflammation in adults with asthma may reflect a counterproductive response to the defective neutrophil microbial killing seen with age, resulting in bystander damage to host airway cells and subsequent mucus hypersecretion and airway remodelling. However, in children with asthma, neutrophils are less associated with adverse features of disease, and it is possible that in children, neutrophils are less pathogenic.In this review, we explore the mechanisms of neutrophil recruitment, changes in cellular function across the life course and the implications this may have for asthma management now and in the future. We also describe the prevalence of neutrophilic asthma globally, with a focus on First Nations people of Australia, New Zealand and North America.

Mitigating Health Risks to Reopen a Clinical Research Laboratory During the COVID-19 Pandemic: A Framework.

BACKGROUND: The COVID-19 pandemic has led to many countries implementing lockdown procedures, resulting in the suspension of laboratory research. With lockdown measures now easing in some areas, many laboratories are preparing to reopen. This is particularly challenging for clinical research laboratories due to the dual risk of patient samples carrying the virus that causes COVID-19, SARS-CoV-2, and the risk to patients being exposed to research staff during clinical sampling. To date, no confirmed transmission of the virus has been confirmed within a laboratory setting; however, operating processes and procedures should be adapted to ensure safe working of samples of positive, negative, or unknown COVID-19 status. OBJECTIVE: In this paper, we propose a framework for reopening a clinical research laboratory and resuming operations with the aim to maximize research capacity while minimizing the risk to research participants and staff. METHODS: This framework was developed by consensus among experienced laboratory staff who have prepared to reopen a clinical research laboratory. RESULTS: Multiple aspects need to be considered to reopen a clinical laboratory. We describe our process to stratify projects by risk, including assessment of donor risk and COVID-19 clinical status, the COVID-19 status of the specific sample type, and how to safely process each sample type. We describe methods to prepare the laboratory for safe working including maintaining social distancing through signage, one-way systems and access arrangements for staff and patients, limiting staff numbers on site and encouraging home working for all nonlaboratory tasks including data analysis and writing. Shared equipment usage was made safe by adapting booking systems to allow for the deployment of cleaning protocols. All risk assessments and standard operating procedures were rewritten and approved by local committees, and staff training was initiated to ensure compliance. CONCLUSIONS: Laboratories can adopt and adapt this framework to expedite reopening a clinical laboratory during the current COVID-19 pandemic while mitigating the risk to research participants and staff.

Frailty Is Associated With Neutrophil Dysfunction Which Is Correctable With Phosphoinositol-3-Kinase Inhibitors.

Neutrophil dysfunction has been described with age, appears exaggerated in infection, with altered phosphoinositol signaling a potential mechanism. However, functional aging is heterogeneous. Frailty is a negative health status and is more common in older adults. We hypothesized that neutrophil migration may be compromised in frailty, associated with the degree of frailty experienced by the older person. We compared measures of frailty, neutrophil function, and systemic inflammation in 40 young and 77 older community-dwelling adults in the United Kingdom. Systemic neutrophils exhibited an age-associated reduction in the accuracy of migration (chemotaxis) which was further blunted with frailty. The degree of migratory inaccuracy correlated with physical (adjusted hand grip strength) and cognitive (Stroop test) markers of frailty. Regression analysis demonstrated that age, Charlson comorbidity index, and frailty index were able to predict neutrophil chemotaxis. Reduced chemotaxis of neutrophils from frail adults could be reversed using selective PI3K inhibitors. Exposure of neutrophils from young adults to plasma from chronically inflamed frail older adults could not recapitulate the migratory deficit in vitro, and there were no relationships with systemic inflammation and neutrophil dysfunction. Frailty exaggerated the neutrophil deficits seen with advanced age but aspects of the frailty-associated deficit in neutrophil function are rescuable and thus potentially form a therapeutic target to improve outcomes from infection in older adults.

A specific proteinase 3 activity footprint in α1-antitrypsin deficiency.

α1-Antitrypsin (α1-AT) deficiency is a risk factor for emphysema due to tissue damage by serine proteases. Neutrophil elastase (NE) has long been considered the enzyme responsible. However, proteinase 3 (PR3) also produces the pathological features of chronic obstructive pulmonary disease (COPD), is present in the same granules in the neutrophil and is inhibited after NE. We developed a specific footprint assay for PR3 activity and assessed its relationship to an NE footprint in α1-AT deficiency. An ELISA was developed for the specific PR3 fibrinogen cleavage site Aα-Val541. Levels were measured in plasma from 239 PiZZ patients, 94 PiSZ patients, 53 nondeficient healthy smokers and 78 individuals with usual COPD. Subjects underwent extensive demographic characterisation including full lung function and lung computed tomography scanning. Aα-Val541 was greater than the NE footprint in all cohorts, consistent with differential activity. Values were highest in the PiZZ α1-AT-deficient patients and correlated with the NE marker Aα-Val360, but were ∼17 times higher than for the NE footprint, consistent with a greater potential contribution to lung damage. Aα-Val541 was related cross-sectionally to the severity of lung disease (forced expiratory volume in 1 s % pred: rs= -0.284; p<0.001) and was sensitive to augmentation therapy, falling from 287.2 to 48.6 nM (p<0.001). An in vivo plasma footprint of PR3 activity is present in greater quantities than an NE footprint in patients with α1-AT deficiency, is sensitive to augmentation therapy and represents a likely biomarker for dose-ranging studies.

Proteinase 3; a potential target in chronic obstructive pulmonary disease and other chronic inflammatory diseases.

Chronic Obstructive Pulmonary Disease (COPD) is a common, multifactorial lung disease which results in significant impairment of patients' health and a large impact on society and health care burden. It is believed to be the result of prolonged, destructive neutrophilic inflammation which results in progressive damage to lung structures. During this process, large quantities of neutrophil serine proteinases (NSPs) are released which initiate the damage and contribute towards driving a persistent inflammatory state.Neutrophil elastase has long been considered the key NSP involved in the pathophysiology of COPD. However, in recent years, a significant role for Proteinase 3 (PR3) in disease development has emerged, both in COPD and other chronic inflammatory conditions. Therefore, there is a need to investigate the importance of PR3 in disease development and hence its potential as a therapeutic target. Research into PR3 has largely been confined to its role as an autoantigen, but PR3 is involved in triggering inflammatory pathways, disrupting cellular signalling, degrading key structural proteins, and pathogen response.This review summarises what is presently known about PR3, explores its involvement particularly in the development of COPD, and indicates areas requiring further investigation.