RESUMEN
BACKGROUND: For patients with acute respiratory distress syndrome, a ventilator is essential to supply oxygen to tissues, but it may also cause lung damage. In this study, we investigated the role of NOX4 using NOX4 knockout (KO) mice and NOX4 inhibitors in a ventilator-induced lung injury (VILI) model. METHODS: Wild-type (WT) male C57BL/6J mice and NOX4 knockout (KO) male mice were divided into five groups: (1) control group; (2) high tidal ventilation (HTV) group: WT mice + HTV ± DMSO; (3) NOX4 KO group; (4) NOX4 KO with HTV group; (5) NOX4 inhibitor group: WT mice + HTV + NOX4 inhibitor. In the VILI model, the supine position was maintained at 24 mL/kg volume, 0 cm H2O PEEP, 100/min respiratory rate, and 0.21 inspired oxygen fraction. In the NOX4 inhibitor group, 50 µL anti-GKT 137831 inhibitor was injected intraperitoneally, 2 h after ventilator use. After 5 h of HTV, mice in the ventilator group were euthanized, and their lung tissues were obtained for further analysis. In addition, the relationship between EphA2 (which is related to lung injury) and NOX4 was investigated using EphA2 KO mice, and NOX4 and EphA2 levels in the bronchoalveolar lavage fluid (BALF) of 38 patients with pneumonia were examined. RESULTS: Cell counts from BALFs were significantly lower in the NOX4 KO with HTV group (p < 0.01) and EphA2 KO with HTV group (p < 0.001) compared to that in the HTV group. In the NOX4 inhibitor group, cell counts and protein concentrations from BALF were significantly lower than those in the HTV group (both, p < 0.001). In the NOX4 KO group and the NOX4 inhibitor group, EphA2 levels were significantly lower than those in the HTV group (p < 0.001). In patients with respiratory disease, NOX4 and EphA2 levels were significantly higher in patients with pneumonia and patients who received ventilator treatment in the intensive care unit. CONCLUSION: In the VILI model with high tidal volume, NOX4 KO, EphA2 KO or monoclonal antibodies attenuated the VILI. NOX4 and EphA2 levels were significantly higher in patients with pneumonia and especially in mechanical ventilated in the ICU. Inhibition of Nox4 is a potential therapeutic target for the prevention and reduction of VILI.
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NADPH Oxidasa 4 , Lesión Pulmonar Inducida por Ventilación Mecánica , Animales , Humanos , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasa 4/genética , NADPH Oxidasa 4/metabolismo , Transducción de Señal , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismoRESUMEN
In this work, medical diagnosis of sepsis was conducted via quantitative analysis of lysophosphatidylcholine 16:0 (LPC 16:0) by using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry based on a parylene-matrix chip. In the first step, specific mass peaks for the diagnosis of sepsis were searched by comparing MALDI-TOF mass spectra of sepsis patient sera with healthy controls and pneumonia patient sera. Two mass peaks at m/z = 496.3 and 518.3 were chosen as those that are specifically different for sepsis sera to compare with healthy controls and pneumonia patient sera. These mass peaks were identified to be protonated and sodium adducts of LPC 16:0 by using tandem mass spectra (MS2 and MS3) of purely synthesized LPC 16:0 and extracted LPC 16:0 from a healthy control and a sepsis patient. In the next step, a standard curve for LPC 16:0 for the quantitative analysis of LPC 16:0 with MALDI-TOF MS based on the parylene-matrix chip was prepared, and the statistical correlation to the LC-MS analysis results was demonstrated by using the Bland-Altman test and Passing-Bablok regression. Finally, MALDI-TOF MS based on the parylene-matrix chip was used for the quantification of LPC 16:0 with sera from patients with severe sepsis and septic shock (n = 143), pneumonia patients (n = 12), and healthy sera (n = 31). The sensitivity and the selectivity of medical diagnosis of sepsis was estimated to be 97.9% and 95.5% by using MALDI-TOF MS based on the parylene-matrix chip, respectively.
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Lisofosfatidilcolinas/sangre , Polímeros/química , Sepsis/diagnóstico , Xilenos/química , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Femenino , Humanos , Límite de Detección , Masculino , Persona de Mediana Edad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The erythropoietin-producing hepatoma (Eph) receptor tyrosine kinase A2 (EphA2) and its ligand, ephrinA1, play a pivotal role in inflammation and tissue injury by modulating the epithelial and endothelial barrier integrity. Therefore, EphA2 receptor may be a potential therapeutic target for modulating ventilator-induced lung injury (VILI). To support this hypothesis, here, we analyzed EphA2/ephrinA1 signaling in the process of VILI and determined the role of EphA2/ephrinA1 signaling in the protective mechanism of prone positioning in a VILI model. Wild-type mice were ventilated with high (24 ml/kg; positive end-expiratory pressure, 0 cm; 5 h) tidal volume in a supine or prone position. Anti-EphA2 receptor antibody or IgG was administered to the supine position group. Injury was assessed by analyzing the BAL fluid, lung injury scoring, and transmission electron microscopy. Lung lysates were evaluated using cytokine/chemokine ELISA and Western blotting of EphA2, ephrinA1, PI3Kγ, Akt, NF-κB, and P70S6 kinase. EphA2/ephrinA1 expression was higher in the supine high tidal volume group than in the control group, but it did not increase upon prone positioning or anti-EphA2 receptor antibody treatment. EphA2 antagonism reduced the extent of VILI and downregulated the expression of PI3Kγ, Akt, NF-κB, and P70S6 kinase. These findings demonstrate that EphA2/ephrinA1 signaling is involved in the molecular mechanism of VILI and that modulation of EphA2/ehprinA1 signaling by prone position or EphA2 antagonism may be associated with the lung-protective effect. Our data provide evidence for EphA2/ehprinA1 as a promising therapeutic target for modulating VILI.
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Pulmón/enzimología , Posición Prona , Receptor EphA2/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/prevención & control , Animales , Anticuerpos/farmacología , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Modelos Animales de Enfermedad , Efrina-A1/metabolismo , Pulmón/efectos de los fármacos , Pulmón/ultraestructura , Masculino , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor EphA2/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Lesión Pulmonar Inducida por Ventilación Mecánica/enzimología , Lesión Pulmonar Inducida por Ventilación Mecánica/patologíaRESUMEN
The role of all-trans retinoic acid (ATRA) in pulmonary fibrosis is relatively unknown, although this metabolite modulates cell differentiation, proliferation, and development. We aimed to evaluate the role of ATRA in bleomycin-induced pulmonary fibrosis, and whether the mechanism involves EphA2-EphrinA1 and PI3K-Akt signaling. We evaluated three groups of mice: a control group (intraperitoneal DMSO injection 3 times weekly after PBS instillation), bleomycin group (intraperitoneal DMSO injection 3 times weekly after bleomycin instillation), and bleomycin + ATRA group (intraperitoneal ATRA injection 3 times weekly after bleomycin instillation). The cell counts and protein concentration in the bronchoalveolar lavage fluid (BALF), changes in histopathology, Ashcroft score, hydroxyproline assay, expression of several signal pathway proteins including EphA2-EphrinA1, and PI3K-Akt, and cytokine levels were compared among the groups. We found that bleomycin significantly increased the protein concentration in the BALF, Ashcroft score in lung tissue, and hydroxyproline contents in lung lysates. Furthermore, bleomycin upregulated EphA2, EphrinA1, PI3K 110γ, Akt, IL-6 and TNF-α. However, administration of ATRA attenuated the upregulation of EphA2-EphrinA1 and PI3K-Akt after bleomycin instillation, and decreased pulmonary fibrosis. In addition, ATRA suppressed IL-6 and TNF-α production induced by bleomycin-induced injury. Collectively, these data suggest that ATRA attenuates bleomycin-induced pulmonary fibrosis by regulating EphA2-EphrinA1 and PI3K-Akt signaling.
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Citocinas/metabolismo , Efrina-A1/metabolismo , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/prevención & control , Receptor EphA2/metabolismo , Tretinoina/administración & dosificación , Animales , Regulación hacia Abajo/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fibrosis Pulmonar/inducido químicamente , Transducción de Señal/efectos de los fármacosRESUMEN
Eph-Ephrin signalling mediates various cellular processes, including vasculogenesis, angiogenesis, cell migration, axon guidance, fluid homoeostasis and repair after injury. Although previous studies have demonstrated that stimulation of the EphA receptor induces increased vascular permeability and inflammatory response in lung injury, the detailed mechanisms of EphA2 signalling are unknown. In the present study, we evaluated the role of EphA2 signalling in mice with lipopolysaccharide (LPS)-induced lung injury. Acute LPS exposure significantly up-regulated EphA2 and EphrinA1 expression. Compared with LPS+IgG mice (IgG instillation after LPS exposure), LPS+EphA2 mAb mice [EphA2 monoclonal antibody (mAb) instillation posttreatment after LPS exposure] had attenuated lung injury and reduced cell counts and protein concentration of bronchoalveolar lavage fluid (BALF). EphA2 mAb posttreatment down-regulated the expression of phosphoinositide 3-kinases (PI3K) 110γ, phospho-Akt, phospho-NF-κB p65, phospho-Src and phospho-S6K in lung lysates. In addition, inhibiting the EphA2 receptor augmented the expression of E-cadherin, which is involved in cell-cell adhesion. Our study identified EphA2 receptor as an unrecognized modulator of several signalling pathways-including PI3K-Akt-NF-kB, Src-NF-κB, E-cadherin and mTOR-in LPS-induced lung injury. These results suggest that EphA2 receptor inhibitors may function as novel therapeutic agents for LPS-induced lung injury.
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Efrina-A1/metabolismo , Lipopolisacáridos/efectos adversos , Lesión Pulmonar/metabolismo , Receptor EphA2/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Humanos , Pulmón/metabolismo , Lesión Pulmonar/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor EphA2/antagonistas & inhibidores , Receptor EphA2/genética , Transducción de SeñalRESUMEN
PURPOSE: Acute kidney injury (AKI) following sepsis is associated with higher mortality; however, reliable biomarkers for AKI development and recovery remain to be elucidated. MATERIALS AND METHODS: Patients with sepsis admitted to the medical intensive care unit (ICU) of Severance Hospital between June 2018 and May 2019 were prospectively analyzed. Patients were divided into those with and without AKI within 48 hours. Patients with septic AKI were subdivided into AKI-recovery and non-recovery groups based on whether their kidney injury recovered within 7 days. RESULTS: A total of 84 patients were enrolled. The baseline creatinine (2.9 mg/dL vs. 0.8 mg/dL vs. 1.2 mg/dL, p<0.001), Charlson Comorbidity Index (4.5 vs. 2.0 vs. 3.0, p=0.002), Sequential Organ Failure Assessment (10.0 vs. 6.5 vs. 8.0, p<0.001), and Acute Physiology and Chronic Health Evaluation II scores (32.0 vs. 21.5 vs. 30.5, p=0.004) were higher in the non-recovery AKI group compared to the non-AKI and AKI-recovery groups. The Kaplan-Meier curves revealed that non-recovery from AKI was associated with lower survival (p<0.001). High-lactate (p≤0.05) and kynurenine levels (p≤0.05) were associated with non-recovery of renal function following AKI. The areas under the curve for predicting non-recovery from AKI were 0.693 and 0.721 for lactate and kynurenine, respectively. The survival rate was lower in the high-kynurenine (p=0.040) and high-lactate (p=0.010) groups. CONCLUSION: The mortality of patients who recovered from AKI was comparable to that of patients without AKI. Lactate and kynurenine could be useful biomarkers for the diagnosis and recovery of AKI following sepsis.
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Lesión Renal Aguda , Sepsis , Humanos , Pronóstico , Quinurenina , Riñón/fisiología , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/etiología , Biomarcadores , Unidades de Cuidados Intensivos , Sepsis/complicaciones , Sepsis/diagnóstico , Lactatos , Estudios RetrospectivosRESUMEN
Variants in SLC26A4 (pendrin) are the most common reasons for genetic hearing loss and vestibular dysfunction in East Asians. In patients with Pendred syndrome and DFNB4 (autosomal recessive type of genetic hearing loss 4), caused by variants in SLC26A4, the hearing function is residual at birth and deteriorates over several years, with no curative treatment for these disorders. In the present study, we revealed that a novel small molecule restores the expression and function of mutant pendrin. High-throughput screening of 54,000 small molecules was performed. We observed that pendrin corrector (PC2-1) increased the surface expression and anion exchange activity of p.H723R pendrin (H723R-PDS), the most prevalent genetic variant that causes Pendred syndrome and DFNB4. Furthermore, in endogenous H723R-PDS-expressing human nasal epithelial cells, PC2-1 significantly increased the surface expression of pendrin. PC2-1 exhibited high membrane permeability in vitro and high micromolar concentrations in the cochlear perilymph in vivo. In addition, neither inhibition of Kv11.1 activity in the human ether-a-go-go-related gene assay nor cell toxicity in the cell proliferation assay was observed at a high PC2-1 concentration (30 µM). These preclinical data support the hypothesis of the druggability of mutant pendrin using the novel corrector molecule PC2-1. In conclusion, PC2-1 may be a new therapeutic molecule for ameliorating hearing loss and treating vestibular disorders in patients with Pendred syndrome or DFNB4.
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Sepsis is predominantly initiated by bacterial infection and can cause systemic inflammation, which frequently leads to rapid death of the patient. However, this acute systemic inflammatory response requires further investigation from the perspectives of clinical judgment criteria and early treatment strategies for the relief of symptoms. Lysophosphatidylcholine (LPC) 18:0 may relieve septic symptoms, but the relevant mechanism is not clearly understood. Therefore, we aimed to assess the effectiveness of LPC as a therapeutic treatment for acute inflammation in the lung induced by lipopolysaccharide in mice. Systemic inflammation of mice was induced by lipopolysaccharide (LPS) inoculation to investigate the role of LPC in the migration and the immune response of neutrophils during acute lung injury. By employing two-photon intravital imaging of the LPS-stimulated LysM-GFP mice and other in vitro and in vivo assays, we examined whether LPC alleviates the inflammatory effect of sepsis. We also tested the effect of LPC to human neutrophils from healthy control and sepsis patients. Our data showed that LPC treatment reduced the infiltration of innate immune cells into the lung. Specifically, LPC altered neutrophil migratory patterns and enhanced phagocytic efficacy in the damaged lung. Moreover, LPC treatment reduced the release of neutrophil extracellular trap (NET), which can damage tissue in the inflamed organ and exacerbate disease. It also reduced human neutrophil migration under inflammatory environment. Our results suggest that LPC can alleviate sepsis-induced lung inflammation by regulating the function of neutrophils. These findings provide evidence for the beneficial application of LPC treatment as a potential therapeutic strategy for sepsis.
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Under ER stress conditions, the ER form of transmembrane proteins can reach the plasma membrane via a Golgi-independent unconventional protein secretion (UPS) pathway. However, the targeting mechanisms of membrane proteins for UPS are unknown. Here, this study reports that TMED proteins play a critical role in the ER stress-associated UPS of transmembrane proteins. The gene silencing results reveal that TMED2, TMED3, TMED9 and TMED10 are involved in the UPS of transmembrane proteins, such as CFTR, pendrin and SARS-CoV-2 Spike. Subsequent mechanistic analyses indicate that TMED3 recognizes the ER core-glycosylated protein cargos and that the heteromeric TMED2/3/9/10 complex mediates their UPS. Co-expression of all four TMEDs improves, while each single expression reduces, the UPS and ion transport function of trafficking-deficient ΔF508-CFTR and p.H723R-pendrin, which cause cystic fibrosis and Pendred syndrome, respectively. In contrast, TMED2/3/9/10 silencing reduces SARS-CoV-2 viral release. These results provide evidence for a common role of TMED3 and related TMEDs in the ER stress-associated, Golgi-independent secretion of transmembrane proteins.
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COVID-19 , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Estrés del Retículo Endoplásmico , Glicoproteína de la Espiga del Coronavirus , Transportadores de Sulfato , COVID-19/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Transporte de Proteínas , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Rationale: Pendrin is encoded by SLC26A4 and its mutation leads to congenital hearing loss. Additionally, pendrin is up-regulated in inflammatory airway diseases such as chronic obstructive pulmonary disease, allergic rhinitis, and asthma. In this study, the effects of a novel pendrin inhibitor, YS-01, were investigated in an LPS-induced acute lung injury (ALI) mice model, and the mechanism underlying the effect of YS-01 was examined. Methods: Lipopolysaccharide (LPS, 10 mg/kg) was intranasally instilled in wild type (WT) and pendrin-null mice. YS-01 (10 mg/kg) was administered intra-peritoneally before or after LPS inhalation. Lung injury parameters were assessed in the lung tissue and bronchoalveolar lavage fluid (BALF). Pendrin levels in the BALF of 41 patients with acute respiratory distress syndrome (ARDS) due to pneumonia and 25 control (solitary pulmonary nodule) patients were also measured. Results: LPS instillation induced lung injury in WT mice but not in pendrin-null mice. Pendrin expression was increased by LPS stimulation both in vitro and in vivo. YS-01 treatment dramatically attenuated lung injury and reduced BALF cell counts and protein concentration after LPS instillation in WT mice. Proinflammatory cytokines and NF-κB activation were suppressed by YS-01 treatment in LPS-induced ALI mice. In BALF of patients whose ARDS was caused by pneumonia, pendrin expression was up-regulated compared to that in controls (mean, 24.86 vs. 6.83 ng/mL, P < 0.001). Conclusions: A novel pendrin inhibitor, YS-01, suppressed lung injury in LPS-induced ALI mice and our data provide a new strategy for the treatment of inflammatory airway diseases including sepsis-induced ALI.
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Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Lipopolisacáridos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Transportadores de Sulfato/antagonistas & inhibidores , Lesión Pulmonar Aguda/metabolismo , Anciano , Animales , Líquido del Lavado Bronquioalveolar , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , FN-kappa B/metabolismo , Oxazoles/farmacología , Neumonía/tratamiento farmacológico , Neumonía/metabolismo , Síndrome de Dificultad Respiratoria/inducido químicamente , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Sepsis remains a critical problem with high mortality worldwide, but there is still a lack of reliable biomarkers. We aimed to evaluate the serum lysophosphatidylcholine (LPC) 16:0 as a biomarker of sepsis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Patients admitted to intensive care unit at Severance Hospital from March 2017 through June 2018 were prospectively enrolled. The inclusion criteria were the fulfillment of at least two criteria of systemic inflammatory response syndrome (SIRS) or the presence of sepsis. Of the 127 patients, 14 had non-infectious SIRS, 41 had sepsis, and 72 had septic shock. The mean serum LPC 16:0 concentration (µmol/L) in non-infectious SIRS was significantly higher than in patients with sepsis and septic shock (101.1 vs. 48.92, p < 0.05; 101.1 vs. 25.88, p < 0.001, respectively). The area under the curve (AUC) predicting 28-day mortality using ΔLPC16:0 (D1-D0) levels was 0.7, which was comparable with the APACHE II score (AUC 0.692) and SOFA score (AUC 0.67). Mechanical ventilation, CRRT, lactate, Δ LPC16:0 (D1-D0) less than the cut-off value were significantly associated with 28-day mortality in multivariable analysis. Our results suggest that LPC16:0 could be a useful biomarker for sepsis diagnosis and mortality prediction in ICU patients.
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Lisofosfatidilcolinas/sangre , Sepsis/diagnóstico , Sepsis/mortalidad , Choque Séptico/diagnóstico , Choque Séptico/mortalidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Síndrome de Respuesta Inflamatoria Sistémica/diagnóstico , Síndrome de Respuesta Inflamatoria Sistémica/mortalidad , Anciano , Biomarcadores/sangre , Femenino , Humanos , Unidades de Cuidados Intensivos , Masculino , Valor Predictivo de las Pruebas , Tasa de Supervivencia , Factores de TiempoRESUMEN
BACKGROUND: Transforming growth factor ß (TGF-ß), retinoic acid (RA), p38 mitogen-activated protein kinase (MAPK), and MEK signaling play critical roles in cell differentiation, proliferation, and apoptosis. We investigated the effect of RA and the role of these signaling molecules on the phosphorylation of Smad2/3 (p-Smad2/3) induced by TGF-ß1. METHODS: A549 epithelial cells and CCD-11Lu fibroblasts were incubated and stimulated with or without all-trans RA (ATRA) and TGF-ß1 and with MAPK or MEK inhibitors. The levels of p-Smad2/3 were analyzed by western blotting. For animal models, we studied three experimental mouse groups: control, bleomycin, and bleomycin+ATRA group. Changes in histopathology, lung injury score, and levels of TGF-ß1 and Smad3 were evaluated at 1 and 3 weeks. RESULTS: When A549 cells were pre-stimulated with TGF-ß1 prior to RA treatment, RA completely inhibited the p-Smad2/3. However, when A549 cells were pre-treated with RA prior to TGF-ß1 stimulation, RA did not completely suppress the p-Smad2/3. When A549 cells were pre-treated with MAPK inhibitor, TGF-ß1 failed to phosphorylate Smad2/3. In fibroblasts, p38 MAPK inhibitor suppressed TGF-ß1-induced p-Smad2. In a bleomycin-induced lung injury mouse model, RA decreased the expression of TGF-ß1 and Smad3 at 1 and 3 weeks. CONCLUSION: RA had inhibitory effects on the phosphorylation of Smad induced by TGF-ß1 in vitro, and RA also decreased the expression of TGF-ß1 at 1 and 3 weeks in vivo. Furthermore, pre-treatment with a MAPK inhibitor showed a preventative effect on TGF-ß1/Smad phosphorylation in epithelial cells. As a result, a combination of RA and MAPK inhibitors may suppress the TGF-ß1-induced lung injury and fibrosis.
RESUMEN
BACKGROUND: Diverse microbiota exist in the lower respiratory tract. Although next generation sequencing (NGS) is the most widely used microbiome analysis technique, it is difficult to implement NGS in clinical microbiology laboratories. Therefore, we evaluated the performance of conventional culture methods together with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying microbiota in bronchoalveolar lavage (BAL) fluid. METHODS: BAL fluid samples (n=27) were obtained from patients undergoing diagnostic bronchoscopy for lung mass evaluation. Bacterial and fungal culture was performed with conventional media used in clinical microbiology laboratories. On an average, 20 isolated colonies were picked from each agar plate and identified by MALDI-TOF MS. Microbiome analysis using 16S rRNA NGS was conducted for comparison. RESULTS: Streptococcus spp. and Neisseria spp. were most frequently cultured from the BAL fluid samples. In two samples, Enterobacteriaceae grew predominantly on MacConkey agar. Actinomyces and Veillonella spp. were commonly identified anaerobes; gut bacteria, such as Lactobacillus, Bifidobacterium, and Clostridium, and fungi were also isolated. NGS revealed more diverse bacterial communities than culture, and Prevotella spp. were mainly identified solely by NGS. Some bacteria, such as Staphylococcus spp., Clostridium spp., and Bifidobacterium spp., were identified solely by culture, indicating that culture may be more sensitive for detecting certain bacteria. CONCLUSIONS: Culture and NGS of BAL fluid samples revealed common bacteria with some different microbial communities. Despite some limitations, culture combined with MALDI-TOF MS might play a complementary role in microbiome analysis using 16S rRNA NGS.
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Bacterias/genética , Líquido del Lavado Bronquioalveolar/microbiología , Secuenciación de Nucleótidos de Alto Rendimiento , Microbiota , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Bacterias/aislamiento & purificación , Hongos/genética , Hongos/aislamiento & purificación , Humanos , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/metabolismo , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Eph receptors and ephrin ligands have several functions including angiogenesis, cell migration, axon guidance, fluid homeostasis, oncogenesis, inflammation and injury repair. The EphA2 receptor potentially mediates the regulation of vascular permeability and inflammation in response to lung injury. METHODS: Mice were divided into 3 experimental groups to study the role of EphA2 signaling in the lipopolysaccharide (LPS)-induced lung injury model i.e., IgG+phosphate-buffered saline (PBS) group (IgG instillation before PBS exposure), IgG+LPS group (IgG instillation before LPS exposure) and EphA2 monoclonal antibody (mAb)+LPS group (EphA2 mAb pretreatment before LPS exposure). RESULTS: EphA2 and ephrinA1 were upregulated in LPS-induced lung injury. The lung injury score of the EphA2 mAb+LPS group was lower than that of the IgG+LPS group (4.30±2.93 vs. 11.45±1.20, respectively; p=0.004). Cell counts (EphA2 mAb+LPS: 11.33×10(4)±8.84×10(4) vs. IgG+LPS: 208.0×10(4)±122.6×10(4); p=0.018) and total protein concentrations (EphA2 mAb+LPS: 0.52±0.41 mg/mL vs. IgG+LPS: 1.38±1.08 mg/mL; p=0.192) were decreased in EphA2 mAb+LPS group, as compared to the IgG+LPS group. In addition, EphA2 antagonism reduced the expression of phospho-p85, phosphoinositide 3-kinase 110γ, phospho-Akt, nuclear factor κB, and proinflammatory cytokines. CONCLUSION: This results of the study indicated a role for EphA2-ephrinA1 signaling in the pathogenesis of LPS-induced lung injury. Furthermore, EphA2 antagonism inhibits the phosphoinositide 3-kinase-Akt pathway and attenuates inflammation.