Neutrophil-Derived Peptidyl Arginine Deiminase Activity Contributes to Pulmonary Emphysema by Enhancing Elastin Degradation.

In chronic obstructive pulmonary disease (COPD), inflammation gives rise to protease-mediated degradation of the key extracellular matrix protein, elastin, which causes irreversible loss of pulmonary function. Intervention against proteolysis has met with limited success in COPD, due in part to our incomplete understanding of the mechanisms that underlie disease pathogenesis. Peptidyl arginine deiminase (PAD) enzymes are a known modifier of proteolytic susceptibility, but their involvement in COPD in the lungs of affected individuals is underexplored. In this study, we showed that enzyme isotypes PAD2 and PAD4 are present in primary granules of neutrophils and that cells from people with COPD release increased levels of PADs when compared with neutrophils of healthy control subjects. By examining bronchoalveolar lavage and lung tissue samples of patients with COPD or matched smoking and nonsmoking counterparts with normal lung function, we reveal that COPD presents with markedly increased airway concentrations of PADs. Ex vivo, we established citrullinated elastin in the peripheral airways of people with COPD, and in vitro, elastin citrullination significantly enhanced its proteolytic degradation by serine and matrix metalloproteinases, including neutrophil elastase and matrix metalloprotease-12, respectively. These results provide a mechanism by which neutrophil-released PADs affect lung function decline, indicating promise for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.

Glycoengineered recombinant alpha1-antitrypsin results in comparable in vitro and in vivo activities to human plasma-derived protein.

Alpha-1-antitrypsin (A1AT) is a multifunctional, clinically important, high value therapeutic glycoprotein that can be used for the treatment of many diseases such as alpha-1-antitrypsin deficiency, diabetes, graft-versus-host-disease, cystic fibrosis and various viral infections. Currently, the only FDA-approved treatment for A1AT disorders is intravenous augmentation therapy with human plasma-derived A1AT. In addition to its limited supply, this approach poses a risk of infection transmission, since it uses therapeutic A1AT harvested from donors. To address these issues, we sought to generate recombinant human A1AT (rhA1AT) that is chemically and biologically indistinguishable from its plasma-derived counterpart using glycoengineered Chinese Hamster Ovary (geCHO-L) cells. By deleting nine key genes that are part of the CHO glycosylation machinery and expressing the human ST6GAL1 and A1AT genes, we obtained stable, high producing geCHO-L lines that produced rhA1AT having an identical glycoprofile to plasma-derived A1AT (pdA1AT). Additionally, the rhA1AT demonstrated in vitro activity and in vivo half-life comparable to commercial pdA1AT. Thus, we anticipate that this platform will help produce human-like recombinant plasma proteins, thereby providing a more sustainable and reliable source of therapeutics that are cost-effective and better-controlled with regard to purity, clinical safety and quality.

Examining the approach to medical remediation programmes-an observational study.

BACKGROUND: Remediation of underperforming students is recognised as an important tool in medical education; however, there is no universally agreed approach. AIMS: This study aimed to evaluate the effectiveness of a remediation program for final year medical students who failed their first long case assessment (LCA1) and to compare their academic performance with their peers who passed their first long case assessment. METHODS: The study consisted of two phases. Phase 1 analysed the demographics and academic performance data for the 9% of the class in the remediation group. Phase 2 focused on collecting similar data for the remaining 91% of students in the non-remediation group. Statistical analyses including the Wilcoxon rank sum test and Pearson correlation coefficients were used to compare the groups. RESULTS: Phase 1 showed 88% of students who participated in remediation successfully passed the second long case assessment (LCA2); however, 25% of this cohort ultimately failed the academic year due to poor results in other assessments. Phase 2 results revealed that non-remediation group students scored significantly higher in LCA2 (59.71% vs 52.07%, p < 0.001) compared to their remediation counterparts, despite 19% of them failing this assessment. Non-remediation group students consistently outperformed their remediation group counterparts in formative and summative assessments. Overall, 6.25% of the entire class failed the academic year. CONCLUSION: This study demonstrates the need to focus on overall academic performance to identify struggling students rather than one high stakes exam. Most of the students in the remediation programme ultimately passed LCA2.

Monocyte NLRP3 inflammasome and interleukin-1ß activation modulated by alpha-1 antitrypsin therapy in deficient individuals.

INTRODUCTION: Altered complement component 3 (C3) activation in patients with alpha-1 antitrypsin (AAT) deficiency (AATD) has been reported. To understand the potential impact on course of inflammation, the aim of this study was to investigate whether C3d, a cleavage-product of C3, triggers interleukin (IL)-1ß secretion via activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. The objective was to explore the effect of AAT augmentation therapy in patients with AATD on the C3d/complement receptor 3 (CR3) signalling axis of monocytes and on circulating pro-inflammatory markers. METHODS: Inflammatory mediators were detected in blood from patients with AATD (n=28) and patients with AATD receiving augmentation therapy (n=19). Inflammasome activation and IL-1ß secretion were measured in monocytes of patients with AATD, and following C3d stimulation in the presence or absence of CR3 or NLRP3 inhibitors. RESULTS: C3d acting via CR3 induces NLRP3 and pro-IL-1ß production, and through induction of endoplasmic reticulum (ER) stress and calcium flux, triggers caspase-1 activation and IL-1ß secretion. Treatment of individuals with AATD with AAT therapy results in decreased plasma levels of C3d (3.0±1.2 µg/mL vs 1.3±0.5 µg/mL respectively, p<0.0001) and IL-1ß (115.4±30 pg/mL vs 73.3±20 pg/mL, respectively, p<0.0001), with a 2.0-fold decrease in monocyte NLRP3 protein expression (p=0.0303), despite continued ER stress activation. DISCUSSION: These results provide strong insight into the mechanism of complement-driven inflammation associated with AATD. Although the described variance in C3d and NLRP3 activation decreased post AAT augmentation therapy, results demonstrate persistent C3d and monocyte ER stress, with implications for new therapeutics and clinical practice.

Alpha-1 Antitrypsin Augmentation and the Liver Phenotype of Adults With Alpha-1 Antitrypsin Deficiency (Genotype Pi∗ZZ).

BACKGROUND & AIMS: α1-Antitrypsin (AAT) is a major protease inhibitor produced by hepatocytes. The most relevant AAT mutation giving rise to AAT deficiency (AATD), the 'Pi∗Z' variant, causes harmful AAT protein accumulation in the liver, shortage of AAT in the systemic circulation, and thereby predisposes to liver and lung injury. Although intravenous AAT augmentation constitutes an established treatment of AATD-associated lung disease, its impact on the liver is unknown. METHODS: Liver-related parameters were assessed in a multinational cohort of 760 adults with severe AATD (Pi∗ZZ genotype) and available liver phenotyping, of whom 344 received augmentation therapy and 416 did not. Liver fibrosis was evaluated noninvasively via the serum test AST-to-platelet ratio index and via transient elastography-based liver stiffness measurement. Histologic parameters were compared in 15 Pi∗ZZ adults with and 35 without augmentation. RESULTS: Compared with nonaugmented subjects, augmented Pi∗ZZ individuals displayed lower serum liver enzyme levels (AST 71% vs 75% upper limit of normal, P < .001; bilirubin 49% vs 58% upper limit of normal, P = .019) and lower surrogate markers of fibrosis (AST-to-platelet ratio index 0.34 vs 0.38, P < .001; liver stiffness measurement 6.5 vs 7.2 kPa, P = .005). Among biopsied participants, augmented individuals had less pronounced liver fibrosis and less inflammatory foci but no differences in AAT accumulation were noted. CONCLUSIONS: The first evaluation of AAT augmentation on the Pi∗ZZ-related liver disease indicates liver safety of a widely used treatment for AATD-associated lung disease. Prospective studies are needed to confirm the beneficial effects and to demonstrate the potential efficacy of exogenous AAT in patients with Pi∗ZZ-associated liver disease.

Safety and Reactogenicity of COVID-19 Vaccination in Severe Alpha-1 Antitrypsin Deficiency.

Background: Patients with alpha-1 antitrypsin deficiency (AATD) exhibit dysregulated inflammatory responses and a predilection for autoimmunity. While the adverse event (AE) profiles of COVID-19 vaccines in several chronic inflammatory conditions are now available, safety and tolerability data for patients with severe AATD have yet to be described. The feasibility of coadministering vaccines against COVID-19 and influenza in this population is similarly unclear. Methods: We conducted a prospective study of 170 patients with Pi*ZZ genotype AATD receiving their initial vaccination series with ChAdOx1 nCoV-19 (AstraZeneca). Patients were monitored clinically for AEs over the week that followed their first and second doses. In parallel, we conducted the same assessments in patients with Pi*MM genotype chronic obstructive pulmonary disease (COPD) (n=160) and Pi*MM individuals without lung disease (n=150). The Pi*ZZ cohort was subsequently followed through 2 consecutive mRNA-based booster vaccines (monovalent and bivalent BNT162b2, Pfizer/BioNTech). To assess the safety of combined vaccination against COVID-19 and influenza, the quadrivalent influenza vaccine was administered to participants attending for their second COVID-19 booster vaccination, either on the same day or following a 1-week interval. Results: Pi*ZZ AATD participants did not display increased AEs compared to Pi*MM COPD or Pi*MM non-lung disease controls. Although unexpected and serious vaccine-associated AEs did occur, the majority of AEs experienced across the 3 groups were mild and self-limiting. The AATD demographic at highest risk for AEs (especially systemic and prolonged AEs) was young females. No increase in AE risk was observed in patients with established emphysema, sonographic evidence of liver disease, or in those receiving intravenous augmentation therapy. AE incidence declined sharply following the initial vaccine series. Same-day coadministration of the COVID-19 mRNA bivalent booster vaccine and the annual influenza vaccine did not result in increased AEs compared to sequential vaccines 1 week apart. Conclusions: Despite their pro-inflammatory state, patients with severe AATD are not at increased risk of AEs or serious AEs compared to patients with nonhereditary COPD and patients without lung disease. Same-day coadministration of COVID-19 booster vaccines with the annual influenza vaccine is feasible, safe, and well-tolerated in this population.

Augmentation Therapy for Severe Alpha-1 Antitrypsin Deficiency Improves Survival and Is Decoupled from Spirometric Decline-A Multinational Registry Analysis.

Rationale: Intravenous plasma-purified alpha-1 antitrypsin (IV-AAT) has been used as therapy for alpha-1 antitrypsin deficiency (AATD) since 1987. Previous trials (RAPID and RAPID-OLE) demonstrated efficacy in preserving computed tomography of lung density but no effect on FEV1. This observational study evaluated 615 people with severe AATD from three countries with socialized health care (Ireland, Switzerland, and Austria), where access to standard medical care was equal but access to IV-AAT was not. Objectives: To assess the real-world longitudinal effects of IV-AAT. Methods: Pulmonary function and mortality data were utilized to perform longitudinal analyses on registry participants with severe AATD. Measurements and Main Results: IV-AAT confers a survival benefit in severe AATD (P < 0.001). We uncovered two distinct AATD phenotypes based on an initial respiratory diagnosis: lung index and non-lung index. Lung indexes demonstrated a more rapid FEV1 decline between the ages of 20 and 50 and subsequently entered a plateau phase of minimal decline from 50 onward. Consequentially, IV-AAT had no effect on FEV1 decline, except in patients with a Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 2 lung index. Conclusions: This real-world study demonstrates a survival advantage from IV-AAT. This improved survival is largely decoupled from FEV1 decline. The observation that patients with severe AATD fall into two major phenotypes has implications for clinical trial design where FEV1 is a primary endpoint. Recruits into trials are typically older lung indexes entering the plateau phase and, therefore, unlikely to show spirometric benefits. IV-AAT attenuates spirometric decline in lung indexes in GOLD stage 2, a spirometric group commonly outside current IV-AAT commencement recommendations.

Physical Activity, Exercise Capacity and Sedentary Behavior in People with Alpha-1 Antitrypsin Deficiency: A Scoping Review.

Alpha-1 antitrypsin deficiency (AATD) is a hereditary disorder and a genetic risk factor for chronic obstructive pulmonary disease (COPD). Physical activity (PA) is important for the prevention and treatment of chronic disease. Little is known about PA in people with AATD. Therefore, we aimed to map the research undertaken to improve and/or measure PA, sedentary behaviour (SB) or exercise in people with AATD. Searches were conducted in CINAHL, Medline, EMBASE and clinical trial databases for studies published in 2021. Databases were searched for keywords (physical activity, AATD, exercise, sedentary behavior) as well as synonyms of these terms, which were connected using Boolean operators. The search yielded 360 records; 37 records were included for review. All included studies (n = 37) assessed exercise capacity; 22 studies reported the use of the six-minute walk test, the incremental shuttle walk test and cardiopulmonary exercise testing were reported in three studies each. Other objective measures of exercise capacity included a submaximal treadmill test, the Naughton protocol treadmill test, cycle ergometer maximal test, endurance shuttle walk test, constant cycle work rate test, a peak work rate test and the number of flights of stairs a participant was able to walk without stopping. A number of participant self-reported measures of exercise capacity were noted. Only one study aimed to analyze the effects of an intensive fitness intervention on daily PA. One further study reported on an exercise intervention and objectively measured PA at baseline. No studies measured SB. The assessment of PA and use of PA as an intervention in AATD is limited, and research into SB absent. Future research should measure PA and SB levels in people with AATD and explore interventions to enhance PA in this susceptible population.

Effect of elexacaftor/tezacaftor/ivacaftor on airway and systemic inflammation in cystic fibrosis.

Treatment with elexacaftor/tezacaftor/ivacaftor (ETI) has been shown to improve lung function in people with cystic fibrosis (PWCF). However, its biological effects remain incompletely understood. Here we describe alterations in pulmonary and systemic inflammation in PWCF following initiation of ETI. To address this, we collected spontaneously expectorated sputum and matching plasma from PWCF (n=30) immediately prior to ETI therapy, then again at 3 and 12 months. Within 3 months, PWCF demonstrated reduced activity of neutrophil elastase, proteinase three and cathepsin G, and decreased concentrations of interleukin (IL)-1ß and IL-8 in sputum, accompanied by decreased Pseudomonas burden and restoration of secretory leukoprotease inhibitor levels. Once treated with ETI, all airway inflammatory markers studied in PWCF had reduced to levels found in matched non-CF bronchiectasis controls. In PWCF with advanced disease, ETI resulted in decreased plasma concentrations of IL-6, C-reactive protein and soluble TNF receptor one as well as normalisation of levels of the acute phase protein, alpha-1 antitrypsin. These data clarify the immunomodulatory effects of ETI and underscore its role as a disease modifier.

Comprehensive Clinical Diagnostic Pipelines Reveal New Variants in Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency (AATD) is an underdiagnosed disorder associated with mutations in the SERPINA1 gene encoding alpha-1 antitrypsin (AAT). Severe AATD can manifest as pulmonary emphysema and progressive liver disease. Besides the most common pathogenic variants S (E264V) and Z (E342K), many rarer genetic variants of AAT have been found in patients and in the general population. Here we report a panel of new SERPINA1 variants, including 4 null and 16 missense alleles, identified among a cohort of individuals with suspected AATD whose phenotypic follow-up showed inconclusive or atypical results. Because the pathogenic significance of the missense variants was unclear purely on the basis of clinical data, the integration of computational, biochemical, and cellular studies was used to define the associated risk of disease. Established pathogenicity predictors and structural analysis identified a panel of candidate damaging mutations that were characterized by expression in mammalian cell models. Polymer formation, intracellular accumulation, and secretory efficiency were evaluated experimentally. Our results identified two AAT mutants with a Z-like polymerogenic severe deficiency profile (Smilano and Mcampolongo) and three milder variants (Xsarezzo, Pdublin, and Ctiberias). Overall, the experimentally determined behavior of the variants was in agreement with the pathogenicity scores of the REVEL (an ensemble method for predicting the pathogenicity of rare missense variants) predictor, supporting the utility of this bioinformatic tool in the initial assessment of newly identified amino acid substitutions of AAT. Our study, in addition to describing 20 new SERPINA1 variants, provides a model for a multidisciplinary approach to classification of rare AAT variants and their clinical impact on individuals with rare AATD genotypes.

Alpha-1 antitrypsin deficiency: current therapy and emerging targets.

INTRODUCTION: Alpha1 antitrypsin deficiency (AATD), a common hereditary disorder affecting mainly lungs, liver and skin has been the focus of some of the most exciting therapeutic approaches in medicine in the past 5 years. In this review, we discuss the therapies presently available for the different manifestations of AATD and new therapies in the pipeline. AREAS COVERED: We review therapeutic options for the individual lung, liver and skin manifestations of AATD along with approaches which aim to treat all three. Along with this renewed interest in treating AATD come challenges. How is AAT best delivered to the lung? What is the desired level of AAT in the circulation and lungs which therapeutics should aim to provide? Will treating the liver disease increase the potential for lung disease? Are there treatments to target the underlying genetic defect with the potential to prevent all aspects of AATDrelated disease? EXPERT OPINION: With a relatively small population able to participate in clinical studies, increased awareness and diagnosis of AATD is urgently needed. Better, more sensitive clinical parameters will assist in the generation of acceptable and robust evidence of therapeutic effect for current and emerging treatments.

Altered Serum Alpha1-Antitrypsin Protease Inhibition before and after Clinical Hematopoietic Stem Cell Transplantation: Association with Risk for Non-Relapse Mortality.

α1-Antitrypsin (AAT), an acute-phase reactant not unsimilar to C-reactive protein (CRP), is a serine protease inhibitor that harbors tissue-protective and immunomodulatory attributes. Its concentrations appropriately increase during conditions of extensive tissue injury, and it induces immune tolerance, in part, by inhibiting the enzymatic activity of the inflammatory serine protease, proteinase 3 (PR3). Typically administered to patients with genetic AAT deficiency, AAT treatment was recently shown to improve outcomes in patients with steroid-refractory graft-versus-host disease (GVHD). GVHD represents a grave outcome of allogeneic hematopoietic stem cell transplantation (HSCT), a potentially curative intervention for hematological diseases. The procedure requires radio/chemotherapy conditioning of the prospective marrow recipient, a cytotoxic process that causes vast tissue injury and, in some formats, interferes with liver production of AAT. To date, changes in the functional profile of AAT during allogeneic HSCT, and during the cytotoxic intervention that precedes HSCT, are unknown. The present study followed 53 patients scheduled for allogeneic HSCT (trial registration NCT03188601). Serum samples were tested before and after HSCT for AAT and CRP levels and for intrinsic anti-proteolytic activity. The ex vivo response to clinical-grade AAT was tested on circulating patient leukocytes and on a human epithelial cell line treated with patient sera in a gap closure assay. According to the ex vivo experiments, circulating leukocytes responded to AAT with a favorable immune-regulated profile, and epithelial gap closure was enhanced by AAT in sera from GVHD-free patients but not in sera from patients who developed GVHD. According to serum collected prior to HSCT, non-relapse mortality was reliably predicted by combining three components: AAT and CRP levels and serum anti-proteolytic activity. Taken together, HSCT outcomes are significantly affected by the anti-proteolytic function of circulating AAT, supporting early AAT augmentation therapy for allogeneic HSCT patients.

Targeting of Glycosaminoglycans in Genetic and Inflammatory Airway Disease.

In the lung, glycosaminoglycans (GAGs) are dispersed in the extracellular matrix (ECM) occupying the interstitial space between the capillary endothelium and the alveolar epithelium, in the sub-epithelial tissue and in airway secretions. In addition to playing key structural roles, GAGs contribute to a number of physiologic processes ranging from cell differentiation, cell adhesion and wound healing. Cytokine and chemokine-GAG interactions are also involved in presentation of inflammatory molecules to respective receptors leading to immune cell migration and airway infiltration. More recently, pathophysiological roles of GAGs have been described. This review aims to discuss the biological roles and molecular interactions of GAGs, and their impact in the pathology of chronic airway diseases, such as cystic fibrosis and chronic obstructive pulmonary disease. Moreover, the role of GAGs in respiratory disease has been heightened by the current COVID-19 pandemic. This review underlines the essential need for continued research aimed at exploring the contribution of GAGs in the development of inflammation, to provide a better understanding of their biological impact, as well as leads in the development of new therapeutic agents.

Alpha-1 Antitrypsin Therapy Modifies Neutrophil Adhesion in Patients with Obstructive Lung Disease.

Alpha-1 antitrypsin deficiency (AATD) is characterized by neutrophil-dominated inflammation resulting in emphysema. The cholesterol-rich neutrophil outer plasma membrane plays a central role in adhesion and subsequent transmigration to underlying tissues. This study aimed to investigate mechanisms of increased neutrophil adhesion in AATD and whether alpha-1 antitrypsin (AAT) augmentation therapy abrogates this effect. Plasma and blood neutrophils were donated by healthy controls (n = 20), AATD (n = 30), and AATD patients after AAT augmentation therapy (n = 6). Neutrophil membrane protein expression was investigated using liquid chromatography-tandem mass spectrometry. The effect of once-weekly intravenous AAT augmentation therapy was assessed by calcium fluorometric, µ-calpain, and cell adhesion assays. Decreased neutrophil plasma membrane cholesterol content (P = 0.03), yet increased abundance of integrin α-M (fold change 1.91), integrin α-L (fold change 3.76), and cytoskeletal adaptor proteins including talin-1 (fold change 4.04) were detected on AATD neutrophil plasma membrane fractions. The described inflammatory induced structural changes were a result of a more than twofold increased cytosolic calcium concentration (P = 0.02), leading to significant calcium-dependent µ-calpain activity (3.5-fold change; P = 0.005), resulting in proteolysis of the membrane cholesterol trafficking protein caveolin-1. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased caveolin-1 and membrane cholesterol content (111.8 ± 15.5 vs. 64.18 ± 7.8 µg/2 × 107 cells before and after treatment, respectively; P = 0.02), with concurrent decreased neutrophil integrin expression and adhesion. Results demonstrate an auxiliary benefit of AAT augmentation therapy, evident by a decrease in circulating inflammation and controlled neutrophil adhesion.