RESUMO
BACKGROUND: The primary underlying defect in cystic fibrosis (CF) is disrupted ion transport in epithelia throughout the body. It is unclear if symptoms such as airway hyperreactivity (AHR) and increased airway smooth muscle (ASM) volume in people with CF are due to inherent abnormalities in smooth muscle or are secondary to epithelial dysfunction. Transforming Growth Factor beta 1 (TGFß) is an established genetic modifier of CF lung disease and a known driver of abnormal ASM function. Prior studies have demonstrated that CF mice develop greater AHR, goblet cell hyperplasia, and ASM hypertrophy after pulmonary TGFß exposure. However, the mechanism driving these abnormalities in CF lung disease, specifically the contribution of CFTR loss in ASM, was unknown. METHODS: In this study, mice with smooth muscle-specific loss of CFTR function (Cftrfl/fl; SM-Cre mice) were exposed to pulmonary TGFß. The impact on lung pathology and physiology was investigated through examination of lung mechanics, Western blot analysis, and pulmonary histology. RESULTS: Cftrfl/fl; SM-Cre mice treated with TGFß demonstrated greater methacholine-induced AHR than control mice. However, Cftrfl/fl; SM-Cre mice did not develop increased inflammation, ASM area, or goblet cell hyperplasia relative to controls following TGFß exposure. CONCLUSIONS: These results demonstrate a direct smooth muscle contribution to CF airway obstruction mediated by TGFß. Dysfunction in non-epithelial tissues should be considered in the development of CF therapeutics, including potential genetic therapies.
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Asma , Fibrose Cística , Animais , Camundongos , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Hiperplasia/metabolismo , Hiperplasia/patologia , Músculo Liso/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Importance: Preclinical models suggest dysregulation of the renin-angiotensin system (RAS) caused by SARS-CoV-2 infection may increase the relative activity of angiotensin II compared with angiotensin (1-7) and may be an important contributor to COVID-19 pathophysiology. Objective: To evaluate the efficacy and safety of RAS modulation using 2 investigational RAS agents, TXA-127 (synthetic angiotensin [1-7]) and TRV-027 (an angiotensin II type 1 receptor-biased ligand), that are hypothesized to potentiate the action of angiotensin (1-7) and mitigate the action of the angiotensin II. Design, Setting, and Participants: Two randomized clinical trials including adults hospitalized with acute COVID-19 and new-onset hypoxemia were conducted at 35 sites in the US between July 22, 2021, and April 20, 2022; last follow-up visit: July 26, 2022. Interventions: A 0.5-mg/kg intravenous infusion of TXA-127 once daily for 5 days or placebo. A 12-mg/h continuous intravenous infusion of TRV-027 for 5 days or placebo. Main Outcomes and Measures: The primary outcome was oxygen-free days, an ordinal outcome that classifies a patient's status at day 28 based on mortality and duration of supplemental oxygen use; an adjusted odds ratio (OR) greater than 1.0 indicated superiority of the RAS agent vs placebo. A key secondary outcome was 28-day all-cause mortality. Safety outcomes included allergic reaction, new kidney replacement therapy, and hypotension. Results: Both trials met prespecified early stopping criteria for a low probability of efficacy. Of 343 patients in the TXA-127 trial (226 [65.9%] aged 31-64 years, 200 [58.3%] men, 225 [65.6%] White, and 274 [79.9%] not Hispanic), 170 received TXA-127 and 173 received placebo. Of 290 patients in the TRV-027 trial (199 [68.6%] aged 31-64 years, 168 [57.9%] men, 195 [67.2%] White, and 225 [77.6%] not Hispanic), 145 received TRV-027 and 145 received placebo. Compared with placebo, both TXA-127 (unadjusted mean difference, -2.3 [95% CrI, -4.8 to 0.2]; adjusted OR, 0.88 [95% CrI, 0.59 to 1.30]) and TRV-027 (unadjusted mean difference, -2.4 [95% CrI, -5.1 to 0.3]; adjusted OR, 0.74 [95% CrI, 0.48 to 1.13]) resulted in no difference in oxygen-free days. In the TXA-127 trial, 28-day all-cause mortality occurred in 22 of 163 patients (13.5%) in the TXA-127 group vs 22 of 166 patients (13.3%) in the placebo group (adjusted OR, 0.83 [95% CrI, 0.41 to 1.66]). In the TRV-027 trial, 28-day all-cause mortality occurred in 29 of 141 patients (20.6%) in the TRV-027 group vs 18 of 140 patients (12.9%) in the placebo group (adjusted OR, 1.52 [95% CrI, 0.75 to 3.08]). The frequency of the safety outcomes was similar with either TXA-127 or TRV-027 vs placebo. Conclusions and Relevance: In adults with severe COVID-19, RAS modulation (TXA-127 or TRV-027) did not improve oxygen-free days vs placebo. These results do not support the hypotheses that pharmacological interventions that selectively block the angiotensin II type 1 receptor or increase angiotensin (1-7) improve outcomes for patients with severe COVID-19. Trial Registration: ClinicalTrials.gov Identifier: NCT04924660.
Assuntos
COVID-19 , Receptor Tipo 1 de Angiotensina , Sistema Renina-Angiotensina , Vasodilatadores , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Angiotensina II/metabolismo , Angiotensinas/administração & dosagem , Angiotensinas/uso terapêutico , COVID-19/complicações , COVID-19/mortalidade , COVID-19/fisiopatologia , COVID-19/terapia , Hipóxia/tratamento farmacológico , Hipóxia/etiologia , Hipóxia/mortalidade , Infusões Intravenosas , Ligantes , Oligopeptídeos/administração & dosagem , Oligopeptídeos/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptor Tipo 1 de Angiotensina/administração & dosagem , Receptor Tipo 1 de Angiotensina/uso terapêutico , Sistema Renina-Angiotensina/efeitos dos fármacos , SARS-CoV-2 , Vasodilatadores/administração & dosagem , Vasodilatadores/uso terapêuticoRESUMO
Neutrophil extracellular traps (NETs), a key component of early defense against microbial infection, are also associated with tissue injury. NET composition has been reported to vary with some disease states, but the composition and variability of NETs across many healthy subjects provide a critical comparison that has not been well investigated. We evaluated NETs from twelve healthy subjects of varying ages isolated from multiple blood draws over a three-and-one-half-year period to delineate the variability in extracellular DNA, protein, enzymatic activities, and susceptibility to protease inhibitors. We calculated correlations for NET constituents and loss of human bronchial epithelial barrier integrity, measured by transepithelial electrical resistance, after NET exposure. We found that although there was some variability within the same subject over time, the mean NET total DNA, dsDNA, protein, LDH, neutrophil elastase (NE), and proteinase 3 (PR3) in isolated NETs were consistent across subjects. NET serine protease activity varied considerably within the same donor from day to day. The mean NET cathepsin G and MPO were significantly different across donors. IL-8 > IL-1RA > G-CSF were the most abundant cytokines in NETs. There was no significant difference in the mean concentration or variability of IL-8, IL-1RA, G-CSF, IL-1α, IL-1ß, or TNF-α in different subjects' NETs. NET DNA concentration was correlated with increased NET neutrophil elastase activity and higher NET IL-1RA concentrations. The mean reduction in protease activity by protease inhibitors was significantly different across donors. NET DNA concentration correlated best with reductions in the barrier integrity of human bronchial epithelia. Defining NET concentration by DNA content correlates with other NET components and reductions in NET-driven epithelial barrier dysfunction, suggesting DNA is a reasonable surrogate measurement for these complex structures in healthy subjects.
Assuntos
Armadilhas Extracelulares , Humanos , Voluntários Saudáveis , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-8 , Elastase de Leucócito , Fator Estimulador de Colônias de Granulócitos , DNA , Inibidores de ProteasesRESUMO
OBJECTIVES: Evaluate the safety and efficacy of the Janus kinase (JAK)1/JAK2 inhibitor ruxolitinib in COVID-19-associated acute respiratory distress syndrome requiring mechanical ventilation. DESIGN: Phase 3 randomized, double-blind, placebo-controlled trial Ruxolitinib in Participants With COVID-19-Associated Acute Respiratory Distress Syndrome Who Require Mechanical Ventilation (RUXCOVID-DEVENT; NCT04377620). SETTING: Hospitals and community-based private or group practices in the United States (29 sites) and Russia (4 sites). PATIENTS: Eligible patients were greater than or equal to 12 years old, hospitalized with severe acute respiratory syndrome coronavirus 2 infection, and mechanically ventilated with a Pa o2 /F io2 of less than or equal to 300 mm Hg within 6 hours of randomization. INTERVENTIONS: Patients were randomized 2:2:1 to receive twice-daily ruxolitinib 15 mg, ruxolitinib 5 mg, or placebo, each plus standard therapy. MEASUREMENTS AND MAIN RESULTS: The primary endpoint, 28-day mortality, was tested for each ruxolitinib group versus placebo using a mixed-effects logistic regression model and one-tailed significance test (significance threshold: p < 0.025); no type 1 error was allocated to secondary endpoints. Between May 24, 2020 and December 15, 2020, 211 patients (age range, 24-87 yr) were randomized (ruxolitinib 15/5 mg, n = 77/87; placebo, n = 47). Acute respiratory distress syndrome was categorized as severe in 27% of patients (58/211) at randomization; 90% (190/211) received concomitant steroids. Day-28 mortality was 51% (39/77; 95% CI, 39-62%) for ruxolitinib 15 mg, 53% (45/85; 95% CI, 42-64%) for ruxolitinib 5 mg, and 70% (33/47; 95% CI, 55-83%) for placebo. Neither ruxolitinib 15 mg (odds ratio, 0.46 [95% CI, 0.201-1.028]; one-sided p = 0.029) nor 5 mg (odds ratio, 0.42 [95% CI, 0.171-1.023]; one-sided p = 0.028) significantly reduced 28-day mortality versus placebo. Numerical improvements with ruxolitinib 15 mg versus placebo were observed in secondary outcomes including ventilator-, ICU-, and vasopressor-free days. Rates of overall and serious treatment-emergent adverse events were similar across treatments. CONCLUSIONS: The observed reduction in 28-day mortality rate between ruxolitinib and placebo in mechanically ventilated patients with COVID-19-associated acute respiratory distress syndrome was not statistically significant; however, the trial was underpowered owing to early termination.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19 , Síndrome do Desconforto Respiratório , Humanos , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , COVID-19/complicações , SARS-CoV-2 , Síndrome do Desconforto Respiratório/tratamento farmacológico , Respiração Artificial , Resultado do TratamentoRESUMO
Cystic fibrosis (CF) is a lethal genetic disease characterized by progressive lung damage and airway obstruction. The majority of patients demonstrate airway hyperresponsiveness (AHR), which is associated with more rapid lung function decline. Recent studies in the neonatal CF pig demonstrated airway smooth muscle (ASM) dysfunction. These findings, combined with observed CF transmembrane conductance regulator (CFTR) expression in ASM, suggest that a fundamental defect in ASM function contributes to lung function decline in CF. One established driver of AHR and ASM dysfunction is transforming growth factor (TGF) ß1, a genetic modifier of CF lung disease. Prior studies demonstrated that TGFß exposure in CF mice drives features of CF lung disease, including goblet cell hyperplasia and abnormal lung mechanics. CF mice displayed aberrant responses to pulmonary TGFß, with elevated PI3K signaling and greater increases in lung resistance compared with controls. Here, we show that TGFß drives abnormalities in CF ASM structure and function through PI3K signaling that is enhanced in CFTR-deficient lungs. CF and non-CF mice were exposed intratracheally to an adenoviral vector containing the TGFß1 cDNA, empty vector, or PBS only. We assessed methacholine-induced AHR, bronchodilator response, and ASM area in control and CF mice. Notably, CF mice demonstrated enhanced AHR and bronchodilator response with greater ASM area increases compared with non-CF mice. Furthermore, therapeutic inhibition of PI3K signaling mitigated the TGFß-induced AHR and goblet cell hyperplasia in CF mice. These results highlight a latent AHR phenotype in CFTR deficiency that is enhanced through TGFß-induced PI3K signaling.
Assuntos
Fibrose Cística/enzimologia , Fibrose Cística/fisiopatologia , Fosfatidilinositol 3-Quinases/metabolismo , Hipersensibilidade Respiratória/enzimologia , Hipersensibilidade Respiratória/fisiopatologia , Transdução de Sinais , Fator de Crescimento Transformador beta/efeitos adversos , Agonistas Adrenérgicos beta/farmacologia , Albuterol/farmacologia , Animais , Broncoconstrição/efeitos dos fármacos , Células Caliciformes/patologia , Hiperplasia , Pulmão/fisiopatologia , Camundongos Endogâmicos C57BL , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiopatologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Neutrophil extracellular traps (NETs) provide host defense but can contribute to the pathobiology of diverse human diseases. We sought to determine the extent and mechanism by which NETs contribute to human airway cell inflammation. Primary normal human bronchial epithelial cells (HBEs) grown at air-liquid interface and wild-type (wt)CFBE41o- cells (expressing wtCFTR) were exposed to cell-free NETs from unrelated healthy volunteers for 18 h in vitro. Cytokines were measured in the apical supernatant by Luminex, and the effect on the HBE transcriptome was assessed by RNA sequencing. NETs consistently stimulated IL-8, TNF-α, and IL-1α secretion by HBEs from multiple donors, with variable effects on other cytokines (IL-6, G-CSF, and GM-CSF). Expression of HBE RNAs encoding IL-1 family cytokines, particularly IL-36 subfamily members, was increased in response to NETs. NET exposure in the presence of anakinra [recombinant human IL-1 receptor antagonist (rhIL-1RA)] dampened NET-induced changes in IL-8 and TNF-α proteins as well as IL-36α RNA. rhIL-36RA limited the increase in expression of proinflammatory cytokine RNAs in HBEs exposed to NETs. NETs selectively upregulate an IL-1 family cytokine response in HBEs, which enhances IL-8 production and is limited by rhIL-1RA. The present findings describe a unique mechanism by which NETs may contribute to inflammation in human lung disease in vivo. NET-driven IL-1 signaling may represent a novel target for modulating inflammation in diseases characterized by a substantial NET burden.
Assuntos
Brônquios/citologia , Células Epiteliais/metabolismo , Armadilhas Extracelulares/metabolismo , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Adulto , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Elastase de Leucócito/metabolismo , Peroxidase/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
INTRODUCTION: Cystic fibrosis (CF) is a genetic disorder that results in a multi-organ disease with progressive respiratory decline that ultimately leads to premature death. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for the CFTR anion channel. Established CF treatments target downstream manifestations of the primary genetic defect, including pulmonary and nutritional interventions. Areas covered: CFTR modulators are novel therapies that improve the function of CFTR, and have been approved in the past five years to mitigate the effects of several CF-disease causing mutations. This review summarizes currently approved CFTR modulators and discusses emerging modulator therapies in phase II and III clinical trials described on clinical trials.gov as of April, 2017. Results of relevant trials reported in peer-reviewed journals in Pubmed, scientific conference abstracts and sponsor press releases available as of November, 2017 are included. Expert opinion: The current scope of CF therapeutic development is robust and CFTR modulators have demonstrated significant benefit to patients with specific CFTR mutations. We anticipate that in the future healthcare providers will be faced with a different treatment paradigm, initiating CFTR-directed therapies well before the onset of progressive lung disease.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Desenho de Fármacos , Animais , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Humanos , Terapia de Alvo Molecular , MutaçãoRESUMO
Alveolar epithelial regeneration is essential for resolution of the acute respiratory distress syndrome (ARDS). Although neutrophils have traditionally been considered mediators of epithelial damage, recent studies suggest they promote type II pneumocyte (AT2) proliferation, which is essential for regenerating alveolar epithelium. These studies did not, however, evaluate this relationship in an in vivo model of alveolar epithelial repair following injury. To determine whether neutrophils influence alveolar epithelial repair in vivo, we developed a unilateral acid injury model that creates a severe yet survivable injury with features similar to ARDS. Mice that received injections of the neutrophil-depleting Ly6G antibody had impaired AT2 proliferation 24 and 72 h after acid instillation, which was associated with decreased reepithelialization and increased alveolar protein concentration 72 h after injury. As neutrophil depletion itself may alter the cytokine response, we questioned the contribution of neutrophils to alveolar epithelial repair in neutropenic granulocyte-colony stimulating factor (G-CSF)-/- mice. We found that the loss of G-CSF recapitulated the neutrophil response of Ly6G-treated mice and was associated with defective alveolar epithelial repair, similar to neutrophil-depleted mice, and was reversed by administration of exogenous G-CSF. To approach the mechanisms, we employed an unbiased protein analysis of bronchoalveolar lavage fluid from neutrophil-depleted and neutrophil-replete mice 12 h after inducing lung injury. Pathway analysis identified significant differences in multiple signaling pathways that may explain the differences in epithelial repair. These data emphasize an important link between the innate immune response and tissue repair in which neutrophils promote alveolar epithelial regeneration.
Assuntos
Lesão Pulmonar Aguda/patologia , Células Epiteliais Alveolares/patologia , Epitélio/patologia , Neutrófilos/patologia , Regeneração , Ácidos , Lesão Pulmonar Aguda/induzido quimicamente , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Anticorpos/farmacologia , Líquido da Lavagem Broncoalveolar , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Epitélio/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/deficiência , Fator Estimulador de Colônias de Granulócitos/metabolismo , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Proteômica , Regeneração/efeitos dos fármacos , Síndrome do Desconforto Respiratório/patologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND: Immunocompromised patients who develop sepsis while neutropenic are at high risk for morbidity and mortality; however, it is unknown if neutropenic sepsis is associated with distinct clinical and biological characteristics. METHODS: We conducted a prospective cohort study of patients admitted to the medical intensive care unit of an academic medical center with severe sepsis. Patients were followed for the development of acute respiratory distress syndrome (ARDS), acute kidney injury (AKI), and mortality. Plasma proteins, representing the host inflammatory response, anti-inflammatory response, and endothelial leak were measured in 30 % of subjects. Clinical characteristics and plasma protein concentrations of patients with neutropenia at enrollment were compared to patients without neutropenia. RESULTS: Of 797 subjects enrolled, 103 (13 %) were neutropenic at ICU admission. The neutropenic subjects were more often in shock, admitted from the hospital ward, had higher APACHE III scores, and more likely bacteremic. Neutropenia was an independent risk factor for AKI (RR 1.28; 95 % CI 1.04, 1.57; p = 0.03), but not ARDS (RR 0.90; 95 % CI 0.70, 1.17; p = 0.42) or 30-day mortality (RR 1.05; 95 % CI 0.85, 1.31; p = 0.65). Neutropenic subjects had higher plasma interleukin (IL)-6 (457 vs. 249 pg/ml; p = 0.03), IL-8 (581 vs. 94 pg/ml; p <0.001), and granulocyte colony-stimulating factor (G-CSF) (3624 vs. 99 pg/ml; p <0.001). Angiopoietin-2 and IL-1 receptor antagonist concentrations did not differ between groups. CONCLUSIONS: Neutropenic sepsis is associated with a higher AKI risk and concentrations of inflammatory mediators IL-6, IL-8, and G-CSF relative to non-neutropenic patients. These differences may have implications for future therapies targeting neutropenic sepsis.
Assuntos
Neutropenia/classificação , Sepse/classificação , Sepse/mortalidade , APACHE , Injúria Renal Aguda/epidemiologia , Injúria Renal Aguda/mortalidade , Adulto , Idoso , Angiopoietina-2/análise , Angiopoietina-2/sangue , Biomarcadores/análise , Biomarcadores/sangue , Distribuição de Qui-Quadrado , Estudos de Coortes , Estado Terminal/epidemiologia , Feminino , Fator Estimulador de Colônias de Granulócitos/análise , Fator Estimulador de Colônias de Granulócitos/sangue , Humanos , Unidades de Terapia Intensiva/organização & administração , Unidades de Terapia Intensiva/estatística & dados numéricos , Interleucina-6/análise , Interleucina-6/sangue , Interleucina-8/análise , Interleucina-8/sangue , Interleucinas/análise , Interleucinas/sangue , Masculino , Pessoa de Meia-Idade , Neutropenia/epidemiologia , Neutropenia/mortalidade , Pennsylvania/epidemiologia , Estudos Prospectivos , Receptores de Interleucina-1/análise , Receptores de Interleucina-1/sangue , Síndrome do Desconforto Respiratório/epidemiologia , Síndrome do Desconforto Respiratório/mortalidade , Sepse/epidemiologiaRESUMO
BACKGROUND: Transcatheter aortic valve replacement (TAVR) with the balloon-expandable Sapien transcatheter heart valve improves survival compared to standard therapy in patients with severe aortic stenosis (AS) and is noninferior to surgical aortic valve replacement (AVR) in patients at high operative risk. Nonetheless, a significant proportion of patients may require pre-emptive or emergent support with cardiopulmonary bypass (CPB) and/or intra-aortic balloon pump (IABP) during TAVR due to pre-existing comorbid conditions or as a result of procedural complications. OBJECTIVES: We hypothesized that patients who required CPB or IABP would have increased periprocedural complications and reduced long-term survival. In addition, we sought to determine whether preprocedural variables could predict the need for CPB and IABP. METHODS: The study population included 2,525 patients in the PARTNER Trial (Cohort A and B) and the continuing access registry (CAR). Patients that received CPB or IABP were compared to patients that did not receive either, and then further divided into those that received support pre-TAVR and those that were placed on support emergently. RESULTS: One-hundred sixty-three patients (6.5%) were placed on CPB and/or IABP. The use of CPB or IABP was associated with higher 1 year mortality (49.1% vs. 21.6%, P < 0.001). In multivariable analysis, utilization of CPB or IABP was an independent predictor of 30 day (HR 6.95) and 1-year (HR 2.56) mortality. Although mortality was highest in emergent cases, mortality was also greater in planned CPB and IABP cases compared with non-CPB/IABP cases (53.3% and 40.3% vs. 21.6%, P < 0.001). CONCLUSIONS: These findings indicate that CPB and IABP use in TAVR portends a poor prognosis and its utilization, particularly in the setting of pre-emptive use, needs reconsideration.
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Estenose da Valva Aórtica/terapia , Valva Aórtica , Cateterismo Cardíaco/mortalidade , Ponte Cardiopulmonar/mortalidade , Implante de Prótese de Valva Cardíaca/mortalidade , Balão Intra-Aórtico/mortalidade , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/fisiopatologia , Estenose da Valva Aórtica/diagnóstico , Estenose da Valva Aórtica/mortalidade , Estenose da Valva Aórtica/fisiopatologia , Cateterismo Cardíaco/efeitos adversos , Cateterismo Cardíaco/instrumentação , Ponte Cardiopulmonar/efeitos adversos , Distribuição de Qui-Quadrado , Comorbidade , Feminino , Próteses Valvulares Cardíacas , Implante de Prótese de Valva Cardíaca/efeitos adversos , Implante de Prótese de Valva Cardíaca/instrumentação , Humanos , Balão Intra-Aórtico/efeitos adversos , Estimativa de Kaplan-Meier , Masculino , Análise Multivariada , Modelos de Riscos Proporcionais , Desenho de Prótese , Sistema de Registros , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do Tratamento , Estados UnidosRESUMO
Introduction: Severe COVID-19 is characterized by cytokine storm, an excessive production of proinflammatory cytokines that contributes to acute lung damage and death. Dexamethasone is routinely used to treat severe COVID-19 and has been shown to reduce patient mortality. However, the mechanisms underlying the beneficial effects of dexamethasone are poorly understood. Methods: We conducted transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients with mild disease, and patients with severe COVID-19 with and without dexamethasone treatment. We then treated healthy donor PBMCs in vitro with dexamethasone and investigated the effects of dexamethasone treatment ion channel abundance (by RT-qPCR and flow cytometry) and function (by electrophysiology, Ca2+ influx measurements and cytokine release) in T cells. Results: We observed that dexamethasone treatment in severe COVID-19 inhibited pro-inflammatory and immune exhaustion pathways, circulating cytotoxic and Th1 cells, interferon (IFN) signaling, genes involved in cytokine storm, and Ca2+ signaling. Ca2+ influx is regulated by Kv1.3 potassium channels, but their role in COVID-19 pathogenesis remains elusive. Kv1.3 mRNA was increased in PBMCs of severe COVID-19 patients, and was significantly reduced in the dexamethasone-treated group. In agreement with these findings, in vitro treatment of healthy donor PBMCs with dexamethasone reduced Kv1.3 abundance in T cells and CD56dimNK cells. Furthermore, functional studies showed that dexamethasone treatment significantly reduced Kv1.3 activity, Ca2+ influx and IFN-g production in T cells. Conclusion: Our findings suggest that dexamethasone attenuates inflammatory cytokine release via Kv1.3 suppression, and this mechanism contributes to dexamethasone-mediated immunosuppression in severe COVID-19.
Assuntos
COVID-19 , Humanos , Leucócitos Mononucleares/metabolismo , Cálcio/metabolismo , Síndrome da Liberação de Citocina/tratamento farmacológico , Tratamento Farmacológico da COVID-19 , Citocinas/metabolismo , Dexametasona/farmacologia , Dexametasona/uso terapêuticoRESUMO
Neutrophil Extracellular Traps (NETs), a key component of early defense against microbial infection, are also associated with tissue injury. NET composition has been reported to vary with some disease states, but the composition and variability of NETs across many healthy subjects provides a critical comparison that has not been well investigated. We evaluated NETs from twelve healthy subjects of varying ages isolated from multiple blood draws over a three and one half-year period to delineate the variability in extracellular DNA, protein, enzymatic activities, and susceptibility to protease inhibitors. We calculated correlations for NET constituents and loss of human bronchial epithelial barrier integrity, measured by transepithelial electrical resistance, after NET exposure. We found that although there was some variability within the same subject over time, the mean numbers of neutrophils, protein, LDH, serine protease activities, and cytokines IL-8, IL-1RA, and G-CSF in isolated NETs were consistent across subjects. Total DNA and double stranded DNA content in NETs were different across donors. NETs had little or no TNFα, IL-17A, or GM-CSF. NET DNA concentration correlated with increased NET neutrophil elastase activity and higher NET IL-1RA concentrations. NET serine protease activity varied considerably within the same donor from day-to-day. Mean response to protease inhibitors was significantly different across donors. NET DNA concentration correlated best with reductions in barrier integrity of human bronchial epithelia. Defining NET concentration by DNA content correlates with other NET components and reductions in NET-driven epithelial barrier dysfunction, suggesting DNA is a reasonable surrogate measurement for these complex structures in healthy subjects.
RESUMO
IL-1 has been associated with acute lung injury (ALI) in both humans and animal models, but further investigation of the precise mechanisms involved is needed, and may identify novel therapeutic targets. To discover the IL-1 mediators essential to the initiation and resolution phases of acute lung inflammation, knockout mice (with targeted deletions for either the IL-1 receptor-1, i.e., Il-1r1(-/-), or the IL-1 receptor antagonist, i.e., Il-1rn(-/-)) were exposed to aerosolized LPS, and indices of lung and systemic inflammation were examined over the subsequent 48 hours. The resultant cell counts, histology, protein, and RNA expression of key cytokines were measured. Il-1r1(-/-) mice exhibited decreased neutrophil influx, particularly at 4 and 48 hours after exposure to LPS, as well as reduced bronchoalveolar lavage (BAL) expression of chemokines and granulocyte colony-stimulating factor (G-CSF). On the contrary, Il-1rn(-/-) mice demonstrated increased BAL neutrophil counts, increased BAL total protein, and greater evidence of histologic injury, all most notably 2 days after LPS exposure. Il-1rn(-/-) mice also exhibited higher peripheral neutrophil counts and greater numbers of granulocyte receptor-1 cells in their bone marrow, potentially reflecting their elevated plasma G-CSF concentrations. Furthermore, IL-17A expression was increased in the BAL and lungs of Il-1rn(-/-) mice after exposure to LPS, likely because of increased numbers of γδ T cells in the Il-1rn(-/-) lungs. Blockade with IL-17A monoclonal antibody before LPS exposure decreased the resultant BAL neutrophil counts and lung G-CSF expression in Il-1rn(-/-) mice, 48 hours after exposure to LPS. In conclusion, Il-1rn(-/-) mice exhibit delayed resolution in acute lung inflammation after exposure to LPS, a process that appears to be mediated via the G-CSF/IL-17A axis.
Assuntos
Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/deficiência , Interleucina-17/metabolismo , Pneumonia/imunologia , Lesão Pulmonar Aguda/sangue , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/metabolismo , Análise de Variância , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Líquido da Lavagem Broncoalveolar/química , Quimiocinas CXC/metabolismo , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos/genética , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-17/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Contagem de Leucócitos , Lipopolissacarídeos/farmacologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Pneumonia/sangue , Pneumonia/metabolismo , Receptores Tipo I de Interleucina-1/deficiência , Receptores Tipo I de Interleucina-1/genéticaRESUMO
Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are prone to venous, cerebrovascular, and coronary thrombi, particularly those with severe coronavirus disease 2019 (COVID-19). The pathogenesis is multifactorial and likely involves proinflammatory cascades, development of coagulopathy, and neutrophil extracellular traps, although further investigations are needed. Elevated levels of D-dimers are common in patients with COVID-19 and cannot be used in isolation to predict venous thromboembolism in people with SARS-CoV-2. If given early in hospital admission, therapeutic-dose heparin improves clinical outcomes in patients with moderate COVID-19. To date, antithrombotics have not improved outcomes in patients with severe COVID-19.
Assuntos
Transtornos da Coagulação Sanguínea , COVID-19 , Trombose , Anticoagulantes/uso terapêutico , COVID-19/complicações , Heparina , Humanos , SARS-CoV-2 , Trombose/etiologiaRESUMO
COVID-19 is a clinical syndrome ranging from mild symptoms to severe pneumonia that often leads to respiratory failure, need for mechanical ventilation, and death. Most of the lung damage is driven by a surge in inflammatory cytokines [interleukin-6, interferon-γ, and granulocyte-monocyte stimulating factor (GM-CSF)]. Blunting this hyperinflammation with immunomodulation may lead to clinical improvement. GM-CSF is produced by many cells, including macrophages and T-cells. GM-CSF-derived signals are involved in differentiation of macrophages, including alveolar macrophages (AMs). In animal models of respiratory infections, the intranasal administration of GM-CSF increased the proliferation of AMs and improved outcomes. Increased levels of GM-CSF have been recently described in patients with COVID-19 compared to healthy controls. While GM-CSF might be beneficial in some circumstances as an appropriate response, in this case the inflammatory response is maladaptive by virtue of being later and disproportionate. The inhibition of GM-CSF signaling may be beneficial in improving the hyperinflammation-related lung damage in the most severe cases of COVID-19. This blockade can be achieved through antagonism of the GM-CSF receptor or the direct binding of circulating GM-CSF. Initial findings from patients with COVID-19 treated with a single intravenous dose of mavrilimumab, a monoclonal antibody binding GM-CSF receptor α, showed oxygenation improvement and shorter hospitalization. Prospective, randomized, placebo-controlled trials are ongoing. Anti-GM-CSF monoclonal antibodies, TJ003234 and gimsilumab, will be tested in clinical trials in patients with COVID-19, while lenzilumab received FDA approval for compassionate use. These trials will help inform whether blunting the inflammatory signaling provided by the GM-CSF axis in COVID-19 is beneficial.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Betacoronavirus/imunologia , Infecções por Coronavirus , Sistemas de Liberação de Medicamentos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Pandemias , Pneumonia Viral , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Animais , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/imunologia , SARS-CoV-2 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Poikiloderma with neutropenia (PN, Clericuzio-type poikiloderma with neutropenia) is a rare autosomal recessive disorder caused by biallelic mutations in the USB1 gene (Alias C16orf57 and MPN1). To date, there have been only 37 reported cases worldwide of this disorder that presents with neutropenia, early onset poikiloderma, respiratory infections, palmo-plantar hyperkeratosis, and skeletal defects. Here we described the generation of human induced pluripotent stem cell lines (PN1 and PN2) from the peripheral blood of a 1-year-old patient using the dox-inducible STEMCCA vector. This patient presented with bacteremia, pneumonia, and neutropenia. Analysis of bone marrow demonstrated normal cellularity with trilineage hematopoiesis and neutropenia.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Neutropenia/metabolismo , Anormalidades da Pele/metabolismo , Linhagem Celular , Humanos , Neutropenia/patologia , Anormalidades da Pele/patologiaRESUMO
The Wnt gene family consists of structurally related genes encoding secreted signaling molecules that have been implicated in many developmental processes, including regulation of cell fate and patterning during embryogenesis. Previously, we found that Wnt signaling is required for primitive or yolk sac-derived-erythropoiesis using the murine embryonic stem cell (ESC) system. Here, we examine the effect of Wnt signaling on the formation of early hematopoietic progenitors derived from human ESCs. The first hematopoietic progenitor cells in the human ESC system express the pan-hematopoietic marker CD41 and the erythrocyte marker, glycophorin A or CD235. We have developed a novel serum-free, feeder-free, adherent differentiation system that can efficiently generate large numbers of CD41+CD235+ cells. We demonstrate that this cell population contains progenitors not just for primitive erythroid and megakaryocyte cells but for the myeloid lineage as well and term this population the primitive common myeloid progenitor (CMP). Treatment of mesoderm-specified cells with Wnt3a led to a loss of hematopoietic colony-forming ability while the inhibition of canonical Wnt signaling with DKK1 led to an increase in the number of primitive CMPs. Canonical Wnt signaling also inhibits the expansion and/or survival of primitive erythrocytes and megakaryocytes, but not myeloid cells, derived from this progenitor population. These findings are in contrast to the role of Wnt signaling during mouse ESC differentiation and demonstrate the importance of the human ESC system in studying species-specific differences in development.
Assuntos
Células-Tronco Embrionárias/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Megacariócitos/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Hematopoese , Células-Tronco Hematopoéticas/citologia , Humanos , Megacariócitos/citologia , CamundongosRESUMO
Neutrophils are essential for maintaining innate immune surveillance under normal conditions, but also represent a major contributor to tissue damage during inflammation. Neutrophil homeostasis is therefore tightly regulated. Cxcr2 plays a critical role in neutrophil homeostasis, as Cxcr2(-/-) mice demonstrate mild neutrophilia and severe neutrophil hyperplasia in the bone marrow. The mechanisms underlying these phenotypes, however, are unclear. We report here that Cxcr2 on murine neutrophils inhibits the IL-17A/G-CSF axis that regulates neutrophil homeostasis. Furthermore, enterocyte-derived Cxcl5 in the gut regulates IL-17/G-CSF levels and contributes to Cxcr2-dependent neutrophil homeostasis. Conversely, G-CSF was required for Cxcl5-dependent regulation of neutrophil homeostasis, and inhibition of IL-17A reduced plasma G-CSF concentrations and marrow neutrophil numbers in both Cxcl5(-/-) and Cxcr2(-/-) mice. Cxcr2(-/-) mice constitutively expressed IL-17A and showed increased numbers of IL-17A-producing cells in the lung, terminal ileum, and spleen. Most IL-17-producing splenocytes were responsive to IL-1ß plus IL-23 in vitro. Depletion of commensal microbes by antibiotic treatment in Cxcr2(-/-) mice markedly decreased IL-17A and G-CSF expression, neutrophilia, and marrow myeloid hyperplasia. These data suggest a critical role for Cxcr2, Cxcl5, and commensal bacteria in regulation of the IL-17/G-CSF axis and neutrophil homeostasis at mucosal sites and have implications for the development of treatments for pathologies resulting from either excessive or ineffective neutrophil responses.