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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.
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Elastina , Neutrófilos , Arginina Deiminasa Proteína-Tipo 2 , Arginina Deiminasa Proteína-Tipo 4 , Proteolisis , Enfermedad Pulmonar Obstructiva Crónica , Enfisema Pulmonar , Humanos , Neutrófilos/inmunología , Elastina/metabolismo , Femenino , Masculino , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Persona de Mediana Edad , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patología , Enfisema Pulmonar/inmunología , Anciano , Arginina Deiminasa Proteína-Tipo 2/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Citrulinación , Desiminasas de la Arginina Proteica/metabolismo , Elastasa de Leucocito/metabolismo , Pulmón/inmunología , Pulmón/patologíaRESUMEN
Elastin is an extracellular matrix protein (ECM) that supports elasticity of the lung, and in patients with chronic obstructive pulmonary disease (COPD) and emphysema, the structural changes that reduce the amount of elastic recoil, lead to loss of pulmonary function. We recently demonstrated that elastin is a target of peptidyl arginine deiminase (PAD) enzyme-induced citrullination, thereby leading to enhanced susceptibility of this ECM protein to proteolysis. The current study aimed to investigate the impact of PAD activity in vivo and furthermore assessed whether pharmacological inhibition of PAD activity protects against pulmonary emphysema. Using a Serpina1a-e knockout mouse model, previously shown to develop inflammation-mediated emphysema, we validated the involvement of PADs in airway disease. In line with emphysema development, intratracheal administration of lipopolysaccharide in combination with PADs provoked significant airspace enlargement (P < 0.001) and diminished lung function, including loss of lung tissue elastance (P = 0.0217) and increases in lung volumes (P = 0.0463). Intraperitoneal treatment of mice with the PAD inhibitor, BB-Cl-amidine, prevented PAD/LPS-mediated lung function decline and emphysema and reduced levels of citrullinated airway elastin (P = 0.0199). These results provide evidence for the impact of PADs on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.
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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.
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Enfermedad Pulmonar Obstructiva Crónica , Deficiencia de alfa 1-Antitripsina , Adulto , Humanos , Persona de Mediana Edad , Adulto Joven , alfa 1-Antitripsina/uso terapéutico , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/complicaciones , Deficiencia de alfa 1-Antitripsina/diagnóstico , Deficiencia de alfa 1-Antitripsina/tratamiento farmacológico , Pulmón , Fenotipo , Sistema de RegistrosRESUMEN
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.
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Fibrosis Quística , Humanos , Fibrosis Quística/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Mutación , Aminofenoles/uso terapéutico , Benzodioxoles/uso terapéuticoRESUMEN
Rationale: Coronavirus disease (COVID-19) is a global threat to health. Its inflammatory characteristics are incompletely understood.Objectives: To define the cytokine profile of COVID-19 and to identify evidence of immunometabolic alterations in those with severe illness.Methods: Levels of IL-1ß, IL-6, IL-8, IL-10, and sTNFR1 (soluble tumor necrosis factor receptor 1) were assessed in plasma from healthy volunteers, hospitalized but stable patients with COVID-19 (COVIDstable patients), patients with COVID-19 requiring ICU admission (COVIDICU patients), and patients with severe community-acquired pneumonia requiring ICU support (CAPICU patients). Immunometabolic markers were measured in circulating neutrophils from patients with severe COVID-19. The acute phase response of AAT (alpha-1 antitrypsin) to COVID-19 was also evaluated.Measurements and Main Results: IL-1ß, IL-6, IL-8, and sTNFR1 were all increased in patients with COVID-19. COVIDICU patients could be clearly differentiated from COVIDstable patients, and demonstrated higher levels of IL-1ß, IL-6, and sTNFR1 but lower IL-10 than CAPICU patients. COVID-19 neutrophils displayed altered immunometabolism, with increased cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, HIF-1α (hypoxia-inducible factor-1α), and lactate. The production and sialylation of AAT increased in COVID-19, but this antiinflammatory response was overwhelmed in severe illness, with the IL-6:AAT ratio markedly higher in patients requiring ICU admission (P < 0.0001). In critically unwell patients with COVID-19, increases in IL-6:AAT predicted prolonged ICU stay and mortality, whereas improvement in IL-6:AAT was associated with clinical resolution (P < 0.0001).Conclusions: The COVID-19 cytokinemia is distinct from that of other types of pneumonia, leading to organ failure and ICU need. Neutrophils undergo immunometabolic reprogramming in severe COVID-19 illness. Cytokine ratios may predict outcomes in this population.
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Reacción de Fase Aguda/inmunología , Proteínas Portadoras/metabolismo , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/metabolismo , Citocinas/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ácido Láctico/metabolismo , Proteínas de la Membrana/metabolismo , Neumonía Viral/inmunología , Neumonía Viral/metabolismo , Hormonas Tiroideas/metabolismo , alfa 1-Antitripsina/inmunología , Reacción de Fase Aguda/metabolismo , Adulto , Anciano , Betacoronavirus , Western Blotting , COVID-19 , Estudios de Casos y Controles , Infecciones Comunitarias Adquiridas/inmunología , Infecciones Comunitarias Adquiridas/metabolismo , Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/fisiopatología , Enfermedad Crítica , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Hospitalización , Humanos , Unidades de Cuidados Intensivos , Interleucina-10/inmunología , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Interleucina-8/inmunología , Tiempo de Internación , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Neutrófilos/metabolismo , Pandemias , Fosforilación , Neumonía/inmunología , Neumonía/metabolismo , Neumonía Viral/mortalidad , Neumonía Viral/fisiopatología , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , SARS-CoV-2 , Índice de Severidad de la Enfermedad , alfa 1-Antitripsina/metabolismo , Proteínas de Unión a Hormona TiroideRESUMEN
Background and Objectives: Alpha-1 antitrypsin is a serine protease inhibitor that demonstrates an array of immunomodulatory functions. Individuals with the genetic condition of alpha-1 antitrypsin deficiency (AATD) are at increased risk of early onset emphysematous lung disease. This lung disease is partly driven by neutrophil mediated lung destruction in an environment of low AAT. As peripheral neutrophil hyper-responsiveness in AATD leads to excessive degranulation and increased migration to the airways, we examined the expression of the membrane voltage-gated proton channel-1 (HVCN1), which is integrally linked to neutrophil function. The objectives of this study were to evaluate altered HVCN1 in AATD neutrophils, serine protease-dependent degradation of HVCN1, and to investigate the ability of serum AAT to control HVCN1 expression. Materials and Methods: Circulating neutrophils were purified from AATD patients (n = 20), AATD patients receiving AAT augmentation therapy (n = 3) and healthy controls (n = 20). HVCN1 neutrophil expression was assessed by flow cytometry and Western blot analysis. Neutrophil membrane bound elastase was measured by fluorescence resonance energy transfer. Results: In this study we demonstrated that HVCN1 protein is under-expressed in AATD neutrophils (p = 0.02), suggesting a link between reduced HVCN1 expression and AAT deficiency. We have demonstrated that HVCN1 undergoes significant proteolytic degradation in activated neutrophils (p < 0.0001), primarily due to neutrophil elastase activity (p = 0.0004). In addition, the treatment of AATD individuals with AAT augmentation therapy increased neutrophil plasma membrane HVCN1 expression (p = 0.01). Conclusions: Our results demonstrate reduced levels of HVCN1 in peripheral blood neutrophils that may influence the neutrophil-dominated immune response in the AATD airways and highlights the role of antiprotease treatment and specifically AAT augmentation therapy in protecting neutrophil membrane expression of HVCN1.
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Neutrófilos , Deficiencia de alfa 1-Antitripsina , Humanos , Pulmón , Proteolisis , Protones , Deficiencia de alfa 1-Antitripsina/tratamiento farmacológico , Deficiencia de alfa 1-Antitripsina/genéticaRESUMEN
INTRODUCTION: Alpha-1 antitrypsin (AAT) deficiency (AATD) is associated with early onset emphysema. The aim of this study was to investigate whether AAT binding to plasma constituents could regulate their activation, and in AATD, exploit this binding event to better understand the condition and uncover novel biomarkers of therapeutic efficacy. METHODS: To isolate AAT linker proteins, plasma samples were separated by size exclusion chromatography, followed by co-immunoprecipitation. AAT binding proteins were identified by mass spectrometry. Complement turnover and activation was determined by ELISA measurement of C3, C3a and C3d levels in plasma of healthy controls (n=15), AATD (n=51), non-AATD patients with obstructive airway disease (n=10) and AATD patients post AAT augmentation therapy (n=5). RESULTS: Direct binding of complement C3 to AAT was identified in vivo and in vitro. Compared with healthy controls, a breakdown product of C3, C3d, was increased in AATD (0.04 µg/mL vs 1.96 µg/mL, p=0.0002), with a significant correlation between radiographic pulmonary emphysema and plasma levels of C3d (R2=0.37, p=0.001). In vivo, AAT augmentation therapy significantly reduced plasma levels of C3d in comparison to patients not receiving AAT therapy (0.15 µg/mL vs 2.18 µg/mL, respectively, p=0.001). DISCUSSION: Results highlight the immune-modulatory impact of AAT on the complement system, involving an important potential role for complement activation in disease pathogenesis in AATD. The association between plasma C3d levels and pulmonary disease severity, that decrease in response to AAT augmentation therapy, supports the exploration of C3d as a candidate biomarker of therapeutic efficacy in AATD.
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Complemento C3/metabolismo , Enfisema Pulmonar/epidemiología , Trastornos Respiratorios/epidemiología , Deficiencia de alfa 1-Antitripsina/epidemiología , Deficiencia de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/uso terapéutico , Anciano , Análisis de Varianza , Biomarcadores/sangre , Western Blotting , Estudios de Casos y Controles , Comorbilidad , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Espectrometría de Masas/métodos , Persona de Mediana Edad , Enfisema Pulmonar/sangre , Enfisema Pulmonar/diagnóstico , Valores de Referencia , Trastornos Respiratorios/sangre , Trastornos Respiratorios/diagnóstico , Índice de Severidad de la Enfermedad , Estadísticas no Paramétricas , Resultado del Tratamiento , Deficiencia de alfa 1-Antitripsina/diagnósticoRESUMEN
Obstructive pulmonary disease in patients with α1 antitrypsin (AAT) deficiency (AATD) occurs earlier in life compared with patients without AATD. To understand this further, the aim of this study was to investigate whether AATD presents with altered neutrophil characteristics, due to the specific lack of plasma AAT, compared with non-AATD COPD.This study focussed on the neutrophil plasma membrane and, by use of label-free tandem mass spectrometry, the proteome of the neutrophil membrane was compared in forced expiratory volume in 1 s (FEV1)-matched AATD, non-AATD COPD and in AATD patients receiving weekly AAT augmentation therapy (n=6 patients per cohort). Altered protein expression in AATD was confirmed by Western blot, ELISA and fluorescence resonance energy transfer analysis.The neutrophil membrane proteome in AATD differed significantly from that of COPD as demonstrated by increased abundance and activity of primary granule proteins including neutrophil elastase on the cell surface in AATD. The signalling mechanism underlying increased degranulation involved Rac2 activation, subsequently resulting in proteinase-activated receptor 2 activation by serine proteinases and enhanced reactive oxygen species production. In vitro and ex vivo, AAT reduced primary granule release and the described plasma membrane variance was resolved post-AAT augmentation therapy in vivo, the effects of which significantly altered the AATD neutrophil membrane proteome to that of a non-AATD COPD cell.These results provide strong insight into the mechanism of neutrophil driven airways disease associated with AATD. Therapeutic AAT augmentation modified the membrane proteome to that of a typical COPD cell, with implications for clinical practice.
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Enfermedad Pulmonar Obstructiva Crónica , Deficiencia de alfa 1-Antitripsina , Volumen Espiratorio Forzado , Humanos , Neutrófilos , Proteoma , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Pruebas de Función Respiratoria , alfa 1-Antitripsina , Deficiencia de alfa 1-Antitripsina/tratamiento farmacológicoRESUMEN
Staphylococcus aureus is an important cause of bovine mastitis, and intramammary infections caused by this pathogen are often characterized as mild, chronic, or persistent. The strains of Staph. aureus associated with mastitis belong to several distinct bovine-adapted bacterial lineages. Studies of host-pathogen interactions have demonstrated that significant differences exist between Staph. aureus strains and lineages in their ability to internalize and to elicit expression of chemokines and pro-inflammatory mediators in bovine cells in vitro. To determine the effect of bacterial strain on the response to intramammary infection in vivo, 14 disease-free, first-lactation cows were randomly allocated to 2 groups and challenged with Staph. aureus strain MOK023 (belonging to CC97) or MOK124 (belonging to CC151). Clinical signs of infection, as well as somatic cell count (SCC), bacterial load, IL-8 and IL-1ß in milk, anti-Staph. aureus IgG in milk and serum, anti-Staph. aureus IgA in milk, and white blood cell populations in milk and blood were monitored for 30 d after the challenge. Cows infected with MOK023 generally developed subclinical mastitis, whereas cows infected with MOK124 generally developed clinical mastitis. Milk yield was reduced to a greater extent in response to infection with MOK124 compared with MOK023 in the first week of the study. Significantly higher SCC, IL-8, and IL-1ß in milk as well as higher anti-Staph. aureus IgG and IgA in milk and anti-Staph. aureus IgG in serum were also observed in response to MOK124 compared with the response to MOK023. Higher proportions of neutrophils were observed in milk of animals infected with MOK124 than in animals infected with MOK023. Higher neutrophil concentration in blood was also observed in the MOK124 group compared with the MOK023 group. Overall, the results indicate that the outcome of mastitis mediated by Staph. aureus is strain dependent.
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Genotipo , Mastitis Bovina/inmunología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/fisiología , Animales , Bovinos , Femenino , Irlanda , Lactancia , Mastitis Bovina/microbiología , Distribución Aleatoria , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genéticaRESUMEN
OBJECTIVES: Resident physicians across disciplines are engaged in teaching at multiple levels. Available literature focuses on medical student education and intra-disciplinary teaching. The national shortage of psychiatrists coupled with an increasing mental illness burden necessitates development of creative interdisciplinary collaboration. The authors report on an interdisciplinary, resident-to-resident didactic series assessing whether such a model could improve internal medicine resident comfort with managing psychiatric illness on inpatient medical wards. METHODS: Internal medicine residents were assessed regarding their comfort level with managing certain common inpatient psychiatric presentations before and after the delivery of a teaching curriculum designed and delivered by psychiatry residents. RESULTS: Internal medicine residents' overall confidence with identifying and managing common psychiatric problems on inpatient medical wards improved. Comfort level with managing depression and demoralization and determining decisional capacity both improved to a statistically significant degree. CONCLUSIONS: Collaborative, interdisciplinary care is complex and its benefits can be difficult to assess. Data from this study showed that interdisciplinary teaching at the resident level has the potential to be an effective means for building collaboration and can lead to a subjective improvement in comfort managing common inpatient psychiatric presentations on medical wards. Additionally, qualitative observations suggest that such an intervention can improve interdisciplinary collaboration.
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Curriculum , Pacientes Internos , Medicina Interna/educación , Internado y Residencia/métodos , Trastornos Mentales/terapia , Psiquiatría/educación , Adulto , Femenino , Humanos , Internado y Residencia/normas , Colaboración Intersectorial , Masculino , Grupo de Atención al PacienteRESUMEN
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.
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Patients with severe alpha-1 antitrypsin deficiency (AATD) are at increased risk for the development of chronic obstructive pulmonary disease (COPD), particularly if they smoke. This, coupled with their predilection for dysregulated inflammation and autoimmunity, makes affected individuals priority candidates for vaccination against coronavirus disease 2019 (COVID-19). To promote vaccine uptake effectively, an understanding of the factors motivating people to proceed with vaccination is essential. The attitudes of patients with AATD towards COVID-19 vaccination have yet to be described. We prospectively studied 170 Pi*ZZ genotype AATD patients, 150 patients with nonhereditary (Pi*MM genotype) COPD and 140 Pi*MM genotype individuals without lung disease receiving first-dose vaccination with ChAdOx1 nCoV-19 (AstraZeneca). Patient attitudes towards vaccination and motivations for getting vaccinated were assessed at the time of the vaccine being offered. Following completion of the 2-dose vaccine series, Pi*ZZ patients were then re-assessed regarding their attitudes towards booster vaccination. The most common primary motivation for accepting vaccination in Pi*ZZ participants ≥50 years old was a fear of illness or death from COVID-19. In contrast, Pi*ZZ patients <50 years most often cited a desire to socialize. The motivation pattern of younger Pi*ZZ AATD patients was similar to that of non-deficient individuals of comparable age, whereas older Pi*ZZ individuals were more closely aligned with Pi*MM COPD and differed from age-matched controls without lung disease. When considering booster vaccination, Pi*ZZ patients were increasingly motivated by a desire to reacquire social freedoms. A desire to reduce the risk of transmission was not a prominent consideration in any of the groups studied. The most commonly cited reason for booster hesitancy was a lack of incentive, given that no additional social freedoms were available to triple-vaccinated individuals compared to those who were double-vaccinated at the time. Taken together, these data may inform policymakers attempting to promote vaccine uptake among patients with AATD.
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Background: Patients with severe coronavirus disease 2019 (COVID-19) develop a febrile pro-inflammatory cytokinemia with accelerated progression to acute respiratory distress syndrome (ARDS). Here we report the results of a phase 2, multicenter, randomized, double-blind, placebo-controlled trial of intravenous (IV) plasma-purified alpha-1 antitrypsin (AAT) for moderate to severe ARDS secondary to COVID-19 (EudraCT 2020-001391-15). Methods: Patients (n = 36) were randomized to receive weekly placebo, weekly AAT (Prolastin, Grifols, S.A.; 120 mg/kg), or AAT once followed by weekly placebo. The primary endpoint was the change in plasma interleukin (IL)-6 concentration at 1 week. In addition to assessing safety and tolerability, changes in plasma levels of IL-1ß, IL-8, IL-10, and soluble tumor necrosis factor receptor 1 (sTNFR1) and clinical outcomes were assessed as secondary endpoints. Findings: Treatment with IV AAT resulted in decreased inflammation and was safe and well tolerated. The study met its primary endpoint, with decreased circulating IL-6 concentrations at 1 week in the treatment group. This was in contrast to the placebo group, where IL-6 was increased. Similarly, plasma sTNFR1 was substantially decreased in the treatment group while remaining unchanged in patients receiving placebo. IV AAT did not definitively reduce levels of IL-1ß, IL-8, and IL-10. No difference in mortality or ventilator-free days was observed between groups, although a trend toward decreased time on ventilator was observed in AAT-treated patients. Conclusions: In patients with COVID-19 and moderate to severe ARDS, treatment with IV AAT was safe, feasible, and biochemically efficacious. The data support progression to a phase 3 trial and prompt further investigation of AAT as an anti-inflammatory therapeutic. Funding: ECSA-2020-009; Elaine Galwey Research Bursary.
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COVID-19 , Síndrome de Dificultad Respiratoria , Deficiencia de alfa 1-Antitripsina , COVID-19/complicaciones , Humanos , Interleucina-10/uso terapéutico , Interleucina-6/uso terapéutico , Interleucina-8/uso terapéutico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , alfa 1-Antitripsina/uso terapéutico , Deficiencia de alfa 1-Antitripsina/tratamiento farmacológicoRESUMEN
BACKGROUND: Interleukin-6 (IL-6) is elevated in SARS-CoV-2 infection. IL-6 regulates acute-phase proteins, such as alpha-1 antitrypsin (AAT), a key lung anti-protease. We investigated the protease-anti-protease balance in the circulation and pulmonary compartments in SARS-CoV-2 acute respiratory distress syndrome (ARDS) compared to non-SARS-CoV-2 ARDS (nsARDS) and the effects of tocilizumab (IL-6 receptor antagonist) on anti-protease defence in SARS-CoV-2 infection. METHODS: Levels and activity of AAT and neutrophil elastase (NE) were measured in plasma, airway tissue and tracheal secretions (TA) of people with SARS-CoV-2 ARDS or nsARDS. AAT and IL-6 levels were evaluated in people with moderate SARS-CoV-2 infection who received standard of care +/- tocilizumab. FINDINGS: AAT plasma levels doubled in SARS-CoV-2 ARDS. In lung parenchyma AAT levels were increased, as was the percentage of neutrophils involved in NET formation. A protease-anti-protease imbalance was detected in TA with active NE and no active AAT. The airway anti-protease, secretory leukoprotease inhibitor was decreased in SARS-CoV-2-infected lungs and cleaved in TA. In nsARDS, plasma AAT levels were elevated but TA samples had less AAT cleavage, with no detectable active NE in most samples. Induction of AAT in ARDS occurred mainly through IL-6. Tocilizumab down-regulated AAT during SARS-CoV-2 infection. INTERPRETATION: There is a protease-anti-protease imbalance in the airways of SARS-CoV-2-ARDS patients. This imbalance is a target for anti-protease therapy. FUNDING: NIH Serological Sciences Network, National Heart, Lung, and Blood Institute and National Institute of Diabetes and Digestive and Kidney Diseases.
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Tratamiento Farmacológico de COVID-19 , Síndrome de Dificultad Respiratoria , Deficiencia de alfa 1-Antitripsina , Humanos , Péptido Hidrolasas , Síndrome de Dificultad Respiratoria/etiología , SARS-CoV-2RESUMEN
For many years, the lung disease associated with alpha-1 antitrypsin (AAT) deficiency (AATD) was perceived as being secondary to an imbalance between this serine protease inhibitor and the target protease, neutrophil elastase (NE). More recently, a greater understanding of the pathways leading to lung inflammation has shed light on new potential attributes and presented AATD as an inflammatory condition in which proteases and neutrophils still play a major role, but in which pro-inflammatory cytokines, either induced by the actions of NE or by other pro-inflammatory processes normally modulated by AAT, are involved. In this review, we will look at the various cytokines centrally involved in AATD lung disease, and how a greater understanding of their contribution may help development of targeted therapies.
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In people with cystic fibrosis (PWCF), inflammation with concurrent infection occurs from a young age and significantly influences lung disease progression. Studies indicate that neutrophils are important effector cells in the pathogenesis of CF and in the development of anti-neutrophil cytoplasmic autoantibodies (ANCA). ANCA specific for bactericidal permeability increasing protein (BPI-ANCA) are detected in people with CF, and correlate with infection with Pseudomonas aeruginosa. The aim of this study was to determine the signaling mechanism leading to increased BPI release by CF neutrophils, while identifying IgG class BPI-ANCA in CF airways samples as the cause for impaired antimicrobial activity of BPI against P. aeruginosa. Plasma and/or bronchoalveolar lavage fluid (BAL) was collected from PWCF (n = 40), CF receiving ivacaftor therapy (n = 10), non-CF patient cohorts (n = 7) and healthy controls (n = 38). Plasma and BAL BPI and BPI-ANCA were measured by ELISA and GTP-bound Rac2 detected using an in vitro assay. The antibacterial effect of all treatments tested was determined by colony forming units enumeration. Levels of BPI are significantly increased in plasma (p = 0.007) and BALF (p < 0.0001) of PWCF. The signaling mechanism leading to increased degranulation and exocytosis of BPI by CF neutrophils (p = 0.02) involved enhancement of Rac2 GTP-loading (p = 0.03). The full-length BPI protein was detectable in all CF BAL samples and patients displayed ANCA with BPI specificity. IgG class autoantibodies were purified from CF BAL complexed to BPI (n=5), with IgG autoantibody cross-linking of antigen preventing BPI induced P. aeruginosa killing (p < 0.0001). Results indicate that the immune-mediated diminished antimicrobial defense, attributed to anti-BPI-IgG, necessitates the formation of a drug/immune complex intermediate that can maintain cytotoxic effects of BPI towards Gram-negative pathogens, with the potential to transform the current treatment of CF airways disease.
RESUMEN
Studies have endeavored to understand the cause for impaired antimicrobial killing by neutrophils of people with cystic fibrosis (PWCF). The aim of this study was to focus on the bacterial phagosome. Possible alterations in degranulation of cytoplasmic granules and changes in pH were assessed. Circulating neutrophils were purified from PWCF (n = 28), PWCF receiving ivacaftor therapy (n = 10), and healthy controls (n = 28). Degranulation was assessed by Western blot analysis and flow cytometry. The pH of phagosomes was determined by use of BCECF-AM-labelled Staphylococcus aureus or SNARF labelled Candida albicans. The antibacterial effect of all treatments tested was determined by colony forming units enumeration. Bacterial killing by CF and healthy control neutrophils were found to differ (p = 0.0006). By use of flow cytometry and subcellular fractionation the kinetics of intraphagosomal degranulation were found to be significantly altered in CF phagosomes, as demonstrated by increased primary granule CD63 (p = 0.0001) and myeloperoxidase (MPO) content (p = 0.03). In contrast, decreased secondary and tertiary granule CD66b (p = 0.002) and decreased hCAP-18 and MMP-9 (p = 0.02), were observed. After 8 min phagocytosis the pH in phagosomes of neutrophils of PWCF was significantly elevated (p = 0.0001), and the percentage of viable bacteria was significantly increased compared to HC (p = 0.002). Results demonstrate that the recorded alterations in phagosomal pH generate suboptimal conditions for MPO related peroxidase, and α-defensin and azurocidine enzymatic killing of Staphylococcus aureus and Pseudomonas aeruginosa. The pattern of dysregulated MPO degranulation (p = 0.02) and prolonged phagosomal alkalinization in CF neutrophils were normalized in vivo following treatment with the ion channel potentiator ivacaftor (p = 0.04). Our results confirm that alterations of circulating neutrophils from PWCF are corrected by CFTR modulator therapy, and raise a question related to possible delayed proton channel activity in CF.
Asunto(s)
Candida albicans/inmunología , Degranulación de la Célula/inmunología , Fibrosis Quística/inmunología , Neutrófilos/inmunología , Fagosomas/inmunología , Staphylococcus aureus/inmunología , Adulto , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Masculino , Neutrófilos/microbiología , Neutrófilos/patología , Fagosomas/microbiología , Fagosomas/patologíaRESUMEN
Mastitis is the most economically important disease affecting dairy cattle worldwide. Staphylococcus aureus is a highly prevalent cause of mastitis, causing infections ranging from sub-clinical to gangrenous. However, the interaction between the genotype of the infecting strain of S. aureus and the host response remains largely uncharacterised. To better understand the variation in presentation and outcomes of S. aureus-mediated bovine mastitis, we studied the interaction of a panel of mastitis isolates from several prominent bovine-associated lineages with bovine mammary epithelial cells (bMEC) and neutrophils. Significant differences in immune gene expression by infected primary or immortalised bMEC, or their elaboration of neutrophil chemoattractants, were observed and were dependent on the lineage of the infecting strain. Differences were also apparent in the invasiveness of S. aureus strains and their ability to survive killing by neutrophils. Our results demonstrate that a range of immune responses occur, suggesting the importance of S. aureus strain in dictating mastitis disease course. S. aureus lineages may therefore have adopted differing strategies for exploitation of the intramammary niche. Consequently, improved diagnosis of infecting lineage may enable better prognosis for S. aureus mastitis and reduce morbidity and economic loss.
Asunto(s)
Genotipo , Especificidad del Huésped/genética , Interacciones Huésped-Patógeno/genética , Mastitis Bovina/inmunología , Staphylococcus aureus/inmunología , Animales , Bovinos , Células Cultivadas , Quimiotaxis/inmunología , Citocinas/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica/inmunología , Granulocitos/inmunología , Granulocitos/metabolismo , Especificidad del Huésped/inmunología , Interacciones Huésped-Patógeno/inmunología , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/microbiología , Glándulas Mamarias Animales/patología , Mastitis Bovina/microbiología , Mastitis Bovina/patología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Cultivo Primario de Células , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
In studying the interaction between bacteria and host cells in vitro, the latter are frequently cultured on commercially available biotic matrices such as Matrigel® or Geltrex®. To avoid contamination, host cells are commonly grown in the presence of antibiotics. However, we present here the finding that cell culture on such a matrix in the presence of antibiotics interferes with the outcome of subsequent infection experiments by virtue of diminished bacterial survival. By comparing outcomes for penicillin-susceptible and resistant strains of Staphylococcus aureus, we show that residual penicillin remains in the matrix despite the antibiotics' withdrawal from culture. Hence, the use of antibiotics should be avoided in this context.
Asunto(s)
Antibacterianos/análisis , Antibacterianos/farmacología , Medios de Cultivo/química , Penicilinas/análisis , Penicilinas/farmacología , Staphylococcus aureus/efectos de los fármacos , Animales , Bovinos , Técnicas de Cultivo de Célula , Células Cultivadas , Células Epiteliales/microbiología , Humanos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/microbiología , Sarcoma Experimental/química , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
Clinical presentation of the chronic, heritable condition cystic fibrosis (CF) is complex, with a diverse range of symptoms often affecting multiple organs with varying severity. The primary source of morbidity and mortality is due to progressive destruction of the airways attributable to chronic inflammation arising from microbial colonisation. Antimicrobial therapy combined with practises to remove obstructive mucopurulent deposits form the cornerstone of current therapy. However, new treatment options are emerging which offer, for the first time, the opportunity to effect remission from the underlying cause of CF. Here, we discuss these therapies, their mechanisms of action, and their successes and failures in order to illustrate the shift in the nature of how CF will likely be managed into the future.