RESUMEN
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.
Asunto(s)
Inflamasomas , Interleucina-1beta , Monocitos , Proteína con Dominio Pirina 3 de la Familia NLR , Deficiencia de alfa 1-Antitripsina , alfa 1-Antitripsina , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Interleucina-1beta/metabolismo , Inflamasomas/metabolismo , Monocitos/metabolismo , Masculino , Femenino , Deficiencia de alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/tratamiento farmacológico , Persona de Mediana Edad , Adulto , Anciano , Transducción de SeñalRESUMEN
Pharmacologic therapy is an integral component in the management of most cardiovascular emergencies. This article reviews the pharmacotherapy involved in the treatment of acute coronary syndromes, acute heart failure, and various arrhythmias. The focus will be to provide practical pearls that can be applied at the bedside in the Emergency Department.
Asunto(s)
Síndrome Coronario Agudo , Insuficiencia Cardíaca , Humanos , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/tratamiento farmacológico , Arritmias Cardíacas , Corazón , Síndrome Coronario Agudo/diagnóstico , Síndrome Coronario Agudo/tratamiento farmacológicoRESUMEN
INTRODUCTION: Small diagnostic tissue samples can be inadequate in testing an expanding list of validated oncogenic driver alterations and fail to reflect intratumour heterogeneity (ITGH) in lung cancer. Liquid biopsies are non-invasive and may better reflect ITGH. Most liquid biopsies are performed in the context of circulating tumour DNA (ctDNA) in plasma but Exhaled Breath Condensate (EBC) shows promise as a lung-specific liquid biopsy. METHODS: In this prospective, proof-of-concept study we carried out targeted Next Generation Sequencing (NGS) on diagnostic tissue samples from 125 patients with lung cancer and compared results to plasma and EBC for 5 oncogenic driver mutations (EGFR, KRAS, PIK3CA, ERBB2, BRAF) using an ultrasensitive PCR technique (UltraSEEK™ Lung Panel on the MassARRAY® System, Agena Bioscience, San Diego, CA, USA). RESULTS: There was a significantly higher failure rate due to unamplifiable DNA in tissue NGS (57/125, 45.6%) compared to plasma (27/125, 21.6%, p < 0.001 and EBC (26/125,20.8%, p ≤ 0.001. Consequently, both plasma and EBC identified higher number of mutations compared to tissue NGS. Specifically, there were significantly higher numbers of mutations detected in EGFR, KRAS and PIK3CA in plasma (p = 9.82 × 10-3, p = 3.14 × 10-5, p = 1.95 × 10-3) and EBC (p = 2.18 × 10-3, p = 2.28 × 10-4,p = 0.016) compared to tissue NGS. There was considerable divergence in mutation profiles between plasma and EBC with 34/76 (44%) mutations detected in plasma and 37/74 (41.89%) in EBC unique to their respective liquid biopsy. CONCLUSIONS: The results suggest that EBC is effective in identifying clinically relevant alterations in patients with lung cancer using UltraSEEK™ and has a potential role as an adjunct to plasma testing.
Asunto(s)
ADN Tumoral Circulante , Neoplasias Pulmonares , Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Receptores ErbB/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Oncogenes , Estudios Prospectivos , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Alpha-1 antitrypsin (AAT) is the canonical serine protease inhibitor of neutrophil-derived proteases and can modulate innate immune mechanisms through its anti-inflammatory activities mediated by a broad spectrum of protein, cytokine, and cell surface interactions. AAT contains a reactive methionine residue that is critical for its protease-specific binding capacity, whereby AAT entraps the protease on cleavage of its reactive centre loop, neutralises its activity by key changes in its tertiary structure, and permits removal of the AAT-protease complex from the circulation. Recently, however, the immunomodulatory role of AAT has come increasingly to the fore with several prominent studies focused on lipid or protein-protein interactions that are predominantly mediated through electrostatic, glycan, or hydrophobic potential binding sites. The aim of this review was to investigate the spectrum of AAT molecular interactions, with newer studies supporting a potential therapeutic paradigm for AAT augmentation therapy in disorders in which a chronic immune response is strongly linked.
Asunto(s)
Apolipoproteínas/metabolismo , Caspasas/metabolismo , Proteínas del Sistema Complemento/metabolismo , Citocinas/metabolismo , alfa 1-Antitripsina/metabolismo , Sitios de Unión/genética , COVID-19/metabolismo , COVID-19/virología , Glicosilación , Humanos , Mutación , Unión Proteica , Dominios Proteicos , SARS-CoV-2/fisiología , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/metabolismoRESUMEN
Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD (n = 88) or non-AATD COPD patients (n = 10) and healthy controls (HC) (n = 40). Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.
RESUMEN
BACKGROUND: In January 2020, the WHO declared the SARS-CoV-2 outbreak a public health emergency; by March 11, a pandemic was declared. To date in Ireland, over 3300 patients have been admitted to acute hospitals as a result of infection with COVID-19. AIMS: This article aims to describe the establishment of a COVID Recovery Service, a multidisciplinary service for comprehensive follow-up of patients with a hospital diagnosis of COVID-19 pneumonia. METHODS: A hybrid model of virtual and in-person clinics was established, supported by a multidisciplinary team consisting of respiratory, critical care, infectious diseases, psychiatry, and psychology services. This model identifies patients who need enhanced follow-up following COVID-19 pneumonia and aims to support patients with complications of COVID-19 and those who require integrated community care. RESULTS: We describe a post-COVID-19 service structure together with detailed protocols for multidisciplinary follow-up. One hundred seventy-four patients were discharged from Beaumont Hospital after COVID-19 pneumonia. Sixty-seven percent were male with a median age (IQR) of 66.5 (51-97). Twenty-two percent were admitted to the ICU for mechanical ventilation, 11% had non-invasive ventilation or high flow oxygen, and 67% did not have specialist respiratory support. Early data suggests that 48% of these patients will require medium to long-term specialist follow-up. CONCLUSIONS: We demonstrate the implementation of an integrated multidisciplinary approach to patients with COVID-19, identifying those with increased physical and mental healthcare needs. Our initial experience suggests that significant physical, psychological, and cognitive impairments may persist despite clinical resolution of the infection.
Asunto(s)
COVID-19/rehabilitación , Atención a la Salud/métodos , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , SARS-CoV-2/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
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
FBXL2 is an important ubiquitin E3 ligase component that modulates inflammatory signaling and cell cycle progression, but its molecular regulation is largely unknown. Here, we show that tumor necrosis factor alpha (TNF-α), a critical cytokine linked to the inflammatory response during skeletal muscle regeneration, suppressed Fbxl2 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of Fbxl2 in skeletal myoblasts resulted in increased proliferative responses characterized by activation of mitogen-activated protein (MAP) kinases and nuclear factor kappa B and decreased myogenic differentiation, as reflected by reduced expression of myogenin and impaired myotube formation. TNF-α did not destabilize the Fbxl2 transcript (half-life [t1/2], â¼10 h) but inhibited SP1 transactivation of its core promoter, localized to bp -160 to +42 within the proximal 5' flanking region of the Fbxl2 gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the Fbxl2 promoter during cellular differentiation, an effect that was less pronounced during proliferation or after TNF-α exposure. TNF-α, via activation of JNK, mediated phosphorylation of SP1 that impaired its binding to the Fbxl2 promoter, resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of Fbxl2 is required for skeletal muscle differentiation, a process that is interrupted by a key proinflammatory myopathic cytokine.IMPORTANCE Skeletal muscle regeneration and repair involve the recruitment and proliferation of resident satellite cells that exit the cell cycle during the process of myogenic differentiation to form myofibers. We demonstrate that the ubiquitin E3 ligase subunit FBXL2 is essential for skeletal myogenesis through its important effects on cell cycle progression and cell proliferative signaling. Further, we characterize a new mechanism whereby sustained stimulation by a major proinflammatory cytokine, TNF-α, regulates skeletal myogenesis by inhibiting the interaction of SP1 with the Fbxl2 core promoter in proliferating myoblasts. Our findings contribute to the understanding of skeletal muscle regeneration through the identification of Fbxl2 as both a critical regulator of myogenic proliferative processes and a susceptible gene target during inflammatory stimulation by TNF-α in skeletal muscle. Modulation of Fbxl2 activity may have relevance to disorders of muscle wasting associated with sustained proinflammatory signaling.
Asunto(s)
Proteínas F-Box/genética , Desarrollo de Músculos , Músculo Esquelético/fisiología , Factor de Transcripción Sp1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células Cultivadas , Humanos , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
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.
Asunto(s)
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
Introduction: Rapid sedation of severely agitated patients is often necessary to ensure the safety of patients and healthcare workers. Intramuscular (IM) ketamine 4-6 mg/kg was previously studied but may carry an increased risk of intubation and other adverse effects. Therefore, the purpose of this case series was to describe the efficacy and safety of a reduced-dose (2 mg/kg) IM ketamine guideline.Methods: Consecutive patients receiving IM ketamine for agitation in the emergency department via our reduced-dose guideline were included. Successful sedation of the agitated patient was defined as documentation from a healthcare provider, a lack of additional sedating medication administration for 30 min following administration of IM ketamine, or ability to complete necessary procedure.Results: Of 15 patients included in this case series, 13 (87%) were adequately sedated with no subsequent intubations due to excess ketamine. The median total dose administered was 157.5 mg and the median weight-based dose was 2 mg/kg. In 11 of the 15 cases, reduced-dose ketamine was used as a second-line agent.Conclusion: Reduced-dose IM ketamine may be effective for severe agitation, particularly when used as a second-line agent.
Asunto(s)
Servicio de Urgencia en Hospital , Hipnóticos y Sedantes/administración & dosificación , Ketamina/administración & dosificación , Agitación Psicomotora/tratamiento farmacológico , Adulto , Anciano , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inyecciones Intramusculares , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Elastin breakdown and the resultant loss of lung elastic recoil is a hallmark of pulmonary emphysema in susceptible individuals as a consequence of tobacco smoke exposure. Systemic alterations to the synthesis and degradation of elastin may be important to our understanding of disease phenotypes in chronic obstructive pulmonary disease. We investigated the association of skin elasticity with pulmonary emphysema, obstructive lung disease, and blood biomarkers of inflammation and tissue protease activity in tobacco-exposed individuals. METHODS: Two hundred and thirty-six Caucasian individuals were recruited into a sub-study of the University of Pittsburgh Specialized Center for Clinically Orientated Research in chronic obstructive pulmonary disease, a prospective cohort study of current and former smokers. The skin viscoelastic modulus (VE), a determinant of skin elasticity, was recorded from the volar forearm and facial wrinkling severity was determined using the Daniell scoring system. RESULTS: In a multiple regression analysis, reduced VE was significantly associated with cross-sectional measurement of airflow obstruction (FEV1/FVC) and emphysema quantified from computed tomography (CT) images, ß = 0.26, p = 0.001 and ß = 0.24, p = 0.001 respectively. In emphysema-susceptible individuals, elasticity-determined skin age was increased (median 4.6 years) compared to the chronological age of subjects without emphysema. Plasma biomarkers of inflammation (TNFR1, TNFR2, CRP, PTX3, and SAA) and matrix metalloproteinase activity (MMP1, TIMP1, TIMP2, and TIMP4) were inversely associated with skin elasticity. CONCLUSIONS: We report that an objective non-invasive determinant of skin elasticity is independently associated with measures of lung function, pulmonary emphysema, and biomarkers of inflammation and tissue proteolysis in tobacco-exposed individuals. Loss of skin elasticity is a novel observation that may link the common pathological processes that drive tissue elastolysis in the extracellular matrix of the skin and lung in emphysema-susceptible individuals.
Asunto(s)
Mediadores de Inflamación/sangre , Metaloproteinasas de la Matriz/sangre , Enfisema Pulmonar/sangre , Envejecimiento de la Piel/patología , Fumadores , Fumar Tabaco/sangre , Anciano , Biomarcadores/sangre , Estudios de Cohortes , Elasticidad/fisiología , Activación Enzimática/fisiología , Femenino , Humanos , Masculino , Estudios Prospectivos , Enfisema Pulmonar/diagnóstico , Método Simple Ciego , Fumar Tabaco/efectos adversosRESUMEN
Rationale: Gene expression of BAL cells, which samples the cellular milieu within the lower respiratory tract, has not been well studied in severe asthma.Objectives: To identify new biomolecular mechanisms underlying severe asthma by an unbiased, detailed interrogation of global gene expression.Methods: BAL cell expression was profiled in 154 asthma and control subjects. Of these participants, 100 had accompanying airway epithelial cell gene expression. BAL cell expression profiles were related to participant (age, sex, race, and medication) and sample traits (cell proportions), and then severity-related gene expression determined by correlating transcripts and coexpression networks to lung function, emergency department visits or hospitalizations in the last year, medication use, and quality-of-life scores.Measurements and Main Results: Age, sex, race, cell proportions, and medications strongly influenced BAL cell gene expression, but leading severity-related genes could be determined by carefully identifying and accounting for these influences. A BAL cell expression network enriched for cAMP signaling components most differentiated subjects with severe asthma from other subjects. Subsequently, an in vitro cellular model showed this phenomenon was likely caused by a robust upregulation in cAMP-related expression in nonsevere and ß-agonist-naive subjects given a ß-agonist before cell collection. Interestingly, ELISAs performed on BAL lysates showed protein levels may partly disagree with expression changes.Conclusions: Gene expression in BAL cells is influenced by factors seldomly considered. Notably, ß-agonist exposure likely had a strong and immediate impact on cellular gene expression, which may not translate to important disease mechanisms or necessarily match protein levels. Leading severity-related genes were discovered in an unbiased, system-wide analysis, revealing new targets that map to asthma susceptibility loci.
Asunto(s)
Asma/genética , Líquido del Lavado Bronquioalveolar/citología , Expresión Génica/genética , Agonistas Adrenérgicos beta/farmacología , Adulto , Asma/metabolismo , Estudios de Casos y Controles , AMP Cíclico/metabolismo , Eosinófilos/metabolismo , Células Epiteliales/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Linfocitos/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Neutrófilos/metabolismo , Análisis de Secuencia de ARN , Índice de Severidad de la Enfermedad , Transducción de Señal/genética , Células THP-1/metabolismoAsunto(s)
Anafilaxia/tratamiento farmacológico , Servicio de Urgencia en Hospital/estadística & datos numéricos , Epinefrina/efectos adversos , Cardiopatías/inducido químicamente , Factores de Edad , Anciano , Anciano de 80 o más Años , Arritmias Cardíacas/inducido químicamente , Presión Sanguínea , Electrocardiografía , Epinefrina/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Troponina/sangreRESUMEN
BACKGROUND: The ubiquitin-proteasome pathway, mediated in part, by ubiquitin E3 ligases, is critical in regulating cellular processes such as cell proliferation, apoptosis, and migration. FBXO17 was recently identified as an F-box protein that targets glycogen synthase kinase-3ß to the E3 ubiquitin ligase protein complex for polyubiquitination and proteasomal degradation. Here, we identified that in several lung adenocarcinoma cell lines, FBXO17 cellular protein was detected at relatively high levels, as was expression in a subset of lung cancers. Hence, we investigated the effects of FBXO17 on cell proliferation. METHODS: Single cell RNA sequencing analysis was performed on a resection of a non-small cell lung carcinoma tumor to examine FBXO17 expression. Multiple lung cancer cell lines were immunoblotted, and The Cancer Genome Atlas was analyzed to determine if FBXO17 expression was amplified in a subset of lung cancers. A549 cells were transfected with empty vector or FBXO17-V5 plasmid and immunoblotted for Akt pathway mediators including PDK1, ERK1/2, ribosomal protein S6, and CREB. Cell proliferation and viability were analyzed by trypan blue exclusion, BrdU incorporation and an MTS-based fluorometric assay. Studies were also performed after transfecting with sifbxo17. Samples were used in an RNA microarray analysis to evaluate pathways affected by reduced FBXO17 gene expression. RESULTS: We observed that overexpression of FBXO17 increased A549 cell proliferation coupled with Akt activation. Ectopically expressed FBXO17 also increased ERK1/2 kinase activation and increased phosphorylation of RPS6, a downstream target of mTOR. We also observed an increased number of cells in S-phase and increased metabolic activity of lung epithelial cells expressing FBXO17. FBXO17 knockdown reduced Akt Ser 473 phosphorylation approaching statistical significance with no effect on Thr 308. However, ERK1/2 phosphorylation, cellular metabolic activity, and overall cell numbers were reduced. When we analyzed RNA profiles of A549 cells with reduced FBXO17 expression, we observed downregulation of several genes associated with cell proliferation and metabolism. CONCLUSIONS: These data support a role for FBXO17 abundance, when left unchecked, in regulating cell proliferation and survival through modulation of Akt and ERK kinase activation. The data raise a potential role for the F-box subunit in modulating tumorigenesis.
Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Proliferación Celular/fisiología , Proteínas F-Box/biosíntesis , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células A549 , Adenocarcinoma del Pulmón/patología , Humanos , Neoplasias Pulmonares/patologíaRESUMEN
We have developed a threedimensional imaging laser radar featuring 3-cm range resolution and single-photon sensitivity. This prototype direct-detection laser radar employs compact, all-solid-state technology for the laser and detector array. The source is a Nd:YAG microchip laser that is diode pumped, passively Q-switched, and frequency doubled. The detector is a gated, passively quenched, two-dimensional array of silicon avalanche photodiodes operating in Geigermode. After describing the system in detail, we present a three-dimensional image, derive performance characteristics, and discuss our plans for future imaging three-dimensional laser radars.