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BACKGROUND: Microbial infection and colonization are frequently associated with disease progression and poor clinical outcomes in bronchiectasis. Identification of pathogen spectrum is crucial for precision treatment at exacerbation of bronchiectasis. METHODS: We conducted a prospective cohort study in patients with bronchiectasis exacerbation onset and stable state. Bronchoalveolar lavage fluid (BALF) was collected for conventional microbiological tests (CMTs) and metagenomic Next-Generation Sequencing (mNGS). Bronchiectasis patients were monitored for documenting the time to the next exacerbation during longitudinal follow-up. RESULTS: We recruited 168 eligible participants in the exacerbation cohorts, and 38 bronchiectasis patients at stable state at longitudinal follow-up. 141 bronchiectasis patients at exacerbation onset had definite or probable pathogens via combining CMTs with mNGS reports. We identified that Pseudomonas aeruginosa, non-tuberculous mycobacteria, Haemophilus influenzae, Nocardia spp, and Staphylococcus aureus were the top 5 pathogens with a higher detection rate in our cohorts via combination of CMTs and mNGS analysis. We also observed strong correlations of Pseudomonas aeruginosa, Haemophilus influenzae, non-tuberculous mycobacteria with disease severity, including the disease duration, Bronchiectasis Severity Index, and lung function. Moreover, the adjusted pathogenic index of potential pathogenic microorganism negatively correlated (r = -0.7280, p < 0.001) with the time to the next exacerbation in bronchiectasis. CONCLUSION: We have revealed the pathogenic microbial spectrum in lower airways and the negative correlation of PPM colonization with the time to the next exacerbation in bronchiectasis. These results suggested that pathogens contribute to the progression of bronchiectasis.
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Bronquiectasia , Humanos , Bronquiectasia/microbiología , Bronquiectasia/diagnóstico , Femenino , Masculino , Estudios Prospectivos , Persona de Mediana Edad , Anciano , Líquido del Lavado Bronquioalveolar/microbiología , Estudios de Cohortes , Estudios de Seguimiento , Adulto , Progresión de la Enfermedad , Estudios LongitudinalesRESUMEN
Background: The early radiological signs of progression in bronchiectasis remain unclear. The objective of the present study was to compare endobronchial optical coherence tomography (EB-OCT) and chest computed tomography (CT) for the evaluation of radiological progression of bronchiectasis via stratification of the presence (TW+) or absence (TW-) of thickened-walled bronchioles surrounding dilated bronchi in patients with bronchiectasis based on CT, and determine the risk factors. Methods: In this prospective cohort study, we performed both chest CT and EB-OCT at baseline and 5-year follow-up, to compare changes in airway calibre metrics. We evaluated bacterial microbiology, sputum matrix metalloproteinase-9 levels and free neutrophil elastase activity at baseline. We compared clinical characteristics and airway calibre metrics between the TW+ and TW- groups. We ascertained radiological progression at 5â years via CT and EB-OCT. Results: We recruited 75 patients between 2014 and 2017. At baseline, EB-OCT metrics (mean luminal diameter (p=0.017), inner airway area (p=0.005) and airway wall area (p=0.009) of seventh- to ninth-generation bronchioles) were significantly greater in the TW+ group than in the TW-group. Meanwhile, EB-OCT did not reveal bronchiole dilatation (compared with the same segment of normal bronchioles) surrounding nondilated bronchi on CT in the TW- group. At 5â years, 53.1% of patients in the TW+ group progressed to have bronchiectasis measured with EB-OCT, compared with only 3.3% in TW- group (p<0.05). 34 patients in the TW+ group demonstrated marked dilatation of medium-sized and small airways. Higher baseline neutrophil elastase activity and TW+ bronchioles on CT predicted progression of bronchiectasis. Conclusion: Thickened-walled bronchioles surrounding the dilated bronchi, identified with EB-OCT, indicates progression of bronchiectasis.
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BACKGROUND: Risk of adverse outcomes in COVID-19 patients by stratifying by the time from symptom onset to confirmed diagnosis status is still uncertain. METHODS: We included 1,590 hospitalized COVID-19 patients confirmed by real-time RT-PCR assay or high-throughput sequencing of pharyngeal and nasal swab specimens from 575 hospitals across China between 11 December 2019 and 31 January 2020. Times from symptom onset to confirmed diagnosis, from symptom onset to first medical visit and from first medical visit to confirmed diagnosis were described and turned into binary variables by the maximally selected rank statistics method. Then, survival analysis, including a log-rank test, Cox regression, and conditional inference tree (CTREE) was conducted, regarding whether patients progressed to a severe disease level during the observational period (assessed as severe pneumonia according to the Chinese Expert Consensus on Clinical Practice for Emergency Severe Pneumonia, admission to an intensive care unit, administration of invasive ventilation, or death) as the prognosis outcome, the dependent variable. Independent factors included whether the time from symptom onset to confirmed diagnosis was longer than 5 days (the exposure) and other demographic and clinical factors as multivariate adjustments. The clinical characteristics of the patients with different times from symptom onset to confirmed diagnosis were also compared. RESULTS: The medians of the times from symptom onset to confirmed diagnosis, from symptom onset to first medical visit, and from first medical visit to confirmed diagnosis were 6, 3, and 2 days. After adjusting for age, sex, smoking status, and comorbidity status, age [hazard ratio (HR): 1.03; 95% CI: 1.01-1.04], comorbidity (HR: 1.84; 95% CI: 1.23-2.73), and a duration from symptom onset to confirmed diagnosis of >5 days (HR: 1.69; 95% CI: 1.10-2.60) were independent predictors of COVID-19 prognosis, which echoed the CTREE models, with significant nodes such as time from symptom onset to confirmed diagnosis, age, and comorbidities. Males, older patients with symptoms such as dry cough/productive cough/shortness of breath, and prior COPD were observed more often in the patients who procrastinated before initiating the first medical consultation. CONCLUSIONS: A longer time from symptom onset to confirmed diagnosis yielded a worse COVID-19 prognosis.
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Importance: Lymphopenia is common and correlates with poor clinical outcomes in patients with coronavirus disease 2019 (COVID-19). Objective: To determine whether a therapy that increases peripheral blood leukocyte and lymphocyte cell counts leads to clinical improvement in patients with COVID-19. Design, Setting and Participants: Between February 18 and April 10, 2020, we conducted an open-label, multicenter, randomized clinical trial at 3 participating centers in China. The main eligibility criteria were pneumonia, a blood lymphocyte cell count of 800 per µL (to convert to ×109/L, multiply by 0.001) or lower, and no comorbidities. Severe acute respiratory syndrome coronavirus 2 infection was confirmed with reverse-transcription polymerase chain reaction testing. Exposures: Usual care alone, or usual care plus 3 doses of recombinant human granulocyte colony-stimulating factor (rhG-CSF, 5 µg/kg, subcutaneously at days 0-2). Main Outcomes and Measures: The primary end point was the time from randomization to improvement of at least 1 point on a 7-category disease severity score. Results: Of 200 participants, 112 (56%) were men and the median (interquartile range [IQR]) age was 45 (40-55) years. There was random assignment of 100 patients (50%) to the rhG-CSF group and 100 (50%) to the usual care group. Time to clinical improvement was similar between groups (rhG-CSF group median of 12 days (IQR, 10-16 days) vs usual care group median of 13 days (IQR, 11-17 days); hazard ratio, 1.28; 95% CI, 0.95-1.71; P = .06). For secondary end points, the proportion of patients progressing to acute respiratory distress syndrome, sepsis, or septic shock was lower in the rhG-CSF group (rhG-CSF group, 2% vs usual care group, 15%; difference, -13%; 95%CI, -21.4% to -5.4%). At 21 days, 2 patients (2%) had died in the rhG-CSF group compared with 10 patients (10%) in the usual care group (hazard ratio, 0.19; 95%CI, 0.04-0.88). At day 5, the lymphocyte cell count was higher in the rhG-CSF group (rhG-CSF group median of 1050/µL vs usual care group median of 620/µL; Hodges-Lehmann estimate of the difference in medians, 440; 95% CI, 380-490). Serious adverse events, such as sepsis or septic shock, respiratory failure, and acute respiratory distress syndrome, occurred in 29 patients (14.5%) in the rhG-CSF group and 42 patients (21%) in the usual care group. Conclusion and Relevance: In preliminary findings from a randomized clinical trial, rhG-CSF treatment for patients with COVID-19 with lymphopenia but no comorbidities did not accelerate clinical improvement, but the number of patients developing critical illness or dying may have been reduced. Larger studies that include a broader range of patients with COVID-19 should be conducted. Trial Registration: Chinese Clinical Trial Registry: ChiCTR2000030007.
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Tratamiento Farmacológico de COVID-19 , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Fármacos Hematológicos/uso terapéutico , Mortalidad Hospitalaria , Linfopenia/tratamiento farmacológico , Corticoesteroides/uso terapéutico , Adulto , Antibacterianos/uso terapéutico , Antivirales/uso terapéutico , Linfocitos B , Recuento de Linfocito CD4 , COVID-19/sangre , COVID-19/complicaciones , COVID-19/fisiopatología , China , Progresión de la Enfermedad , Femenino , Humanos , Células Asesinas Naturales , Recuento de Leucocitos , Recuento de Linfocitos , Linfopenia/sangre , Linfopenia/complicaciones , Masculino , Persona de Mediana Edad , Mortalidad , Ventilación no Invasiva , Terapia por Inhalación de Oxígeno , Proteínas Recombinantes , Síndrome de Dificultad Respiratoria/fisiopatología , Insuficiencia Respiratoria/fisiopatología , SARS-CoV-2 , Sepsis/fisiopatología , Choque Séptico/fisiopatología , Factores de TiempoRESUMEN
BACKGROUND: During the outbreak of coronavirus disease 2019 (COVID-19), consistent and considerable differences in disease severity and mortality rate of patients treated in Hubei province compared to those in other parts of China have been observed. We sought to compare the clinical characteristics and outcomes of patients being treated inside and outside Hubei province, and explore the factors underlying these differences. METHODS: Collaborating with the National Health Commission, we established a retrospective cohort to study hospitalised COVID-19 cases in China. Clinical characteristics, the rate of severe events and deaths, and the time to critical illness (invasive ventilation or intensive care unit admission or death) were compared between patients within and outside Hubei. The impact of Wuhan-related exposure (a presumed key factor that drove the severe situation in Hubei, as Wuhan is the epicentre as well the administrative centre of Hubei province) and the duration between symptom onset and admission on prognosis were also determined. RESULTS: At the data cut-off (31 January 2020), 1590 cases from 575 hospitals in 31 provincial administrative regions were collected (core cohort). The overall rate of severe cases and mortality was 16.0% and 3.2%, respectively. Patients in Hubei (predominantly with Wuhan-related exposure, 597 (92.3%) out of 647) were older (mean age 49.7 versus 44.9â years), had more cases with comorbidity (32.9% versus 19.7%), higher symptomatic burden, abnormal radiologic manifestations and, especially, a longer waiting time between symptom onset and admission (5.7 versus 4.5â days) compared with patients outside Hubei. Patients in Hubei (severe event rate 23.0% versus 11.1%, death rate 7.3% versus 0.3%, HR (95% CI) for critical illness 1.59 (1.05-2.41)) have a poorer prognosis compared with patients outside Hubei after adjusting for age and comorbidity. However, among patients outside Hubei, the duration from symptom onset to hospitalisation (mean 4.4 versus 4.7â days) and prognosis (HR (95%) 0.84 (0.40-1.80)) were similar between patients with or without Wuhan-related exposure. In the overall population, the waiting time, but neither treated in Hubei nor Wuhan-related exposure, remained an independent prognostic factor (HR (95%) 1.05 (1.01-1.08)). CONCLUSION: There were more severe cases and poorer outcomes for COVID-19 patients treated in Hubei, which might be attributed to the prolonged duration of symptom onset to hospitalisation in the epicentre. Future studies to determine the reason for delaying hospitalisation are warranted.
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Infecciones por Coronavirus/mortalidad , Hospitalización , Neumonía Viral/mortalidad , Adulto , Anciano , Betacoronavirus , COVID-19 , Enfermedades Cardiovasculares/epidemiología , China , Estudios de Cohortes , Comorbilidad , Infecciones por Coronavirus/complicaciones , Infecciones por Coronavirus/diagnóstico por imagen , Tos/etiología , Diabetes Mellitus/epidemiología , Brotes de Enfermedades , Disnea/etiología , Fatiga/etiología , Femenino , Fiebre/etiología , Geografía , Humanos , Hipertensión/epidemiología , Unidades de Cuidados Intensivos/estadística & datos numéricos , Pulmón/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Pandemias , Faringitis/etiología , Neumonía Viral/complicaciones , Neumonía Viral/diagnóstico por imagen , Pronóstico , Modelos de Riesgos Proporcionales , Respiración Artificial/estadística & datos numéricos , Estudios Retrospectivos , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Factores de Tiempo , Tiempo de Tratamiento/estadística & datos numéricos , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: The coronavirus disease 2019 (COVID-19) outbreak is evolving rapidly worldwide. OBJECTIVE: To evaluate the risk of serious adverse outcomes in patients with COVID-19 by stratifying the comorbidity status. METHODS: We analysed data from 1590 laboratory confirmed hospitalised patients from 575 hospitals in 31 provinces/autonomous regions/provincial municipalities across mainland China between 11 December 2019 and 31 January 2020. We analysed the composite end-points, which consisted of admission to an intensive care unit, invasive ventilation or death. The risk of reaching the composite end-points was compared according to the presence and number of comorbidities. RESULTS: The mean age was 48.9â years and 686 (42.7%) patients were female. Severe cases accounted for 16.0% of the study population. 131 (8.2%) patients reached the composite end-points. 399 (25.1%) reported having at least one comorbidity. The most prevalent comorbidity was hypertension (16.9%), followed by diabetes (8.2%). 130 (8.2%) patients reported having two or more comorbidities. After adjusting for age and smoking status, COPD (HR (95% CI) 2.681 (1.424-5.048)), diabetes (1.59 (1.03-2.45)), hypertension (1.58 (1.07-2.32)) and malignancy (3.50 (1.60-7.64)) were risk factors of reaching the composite end-points. The hazard ratio (95% CI) was 1.79 (1.16-2.77) among patients with at least one comorbidity and 2.59 (1.61-4.17) among patients with two or more comorbidities. CONCLUSION: Among laboratory confirmed cases of COVID-19, patients with any comorbidity yielded poorer clinical outcomes than those without. A greater number of comorbidities also correlated with poorer clinical outcomes.
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Betacoronavirus , Infecciones por Coronavirus/epidemiología , Neumonía Viral/epidemiología , Adulto , COVID-19 , China/epidemiología , Comorbilidad , Infecciones por Coronavirus/diagnóstico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/diagnóstico , Pronóstico , Factores de Riesgo , SARS-CoV-2RESUMEN
A crucial component of nonalcoholic fatty liver disease (NAFLD) pathogenesis is lipid stress, which may contribute to hepatic inflammation and activation of innate immunity in the liver. However, little is known regarding how dietary lipids, including fat and cholesterol, may facilitate innate immune activation in vivo. We hypothesized that dietary fat and cholesterol drive NAFLD progression to steatohepatitis and hepatic fibrosis by altering the transcription and phenotype of hepatic macrophages. This hypothesis was tested by using RNA-sequencing methods to characterize and analyze sort-purified hepatic macrophage populations that were isolated from mice fed diets with varying amounts of fat and cholesterol. The addition of cholesterol to a high-fat diet triggered hepatic pathology reminiscent of advanced nonalcoholic steatohepatitis (NASH) in humans characterized by signs of cholesterol dysregulation, generation of oxidized low-density lipoprotein, increased recruitment of hepatic macrophages, and significant fibrosis. RNA-sequencing analyses of hepatic macrophages in this model revealed that dietary cholesterol induced a tissue repair and regeneration phenotype in Kupffer cells (KCs) and recruited infiltrating macrophages to a greater degree than fat. Furthermore, comparison of diseased KCs and infiltrating macrophages revealed that these two macrophage subsets are transcriptionally diverse. Finally, direct stimulation of murine and human macrophages with oxidized low-density lipoprotein recapitulated some of the transcriptional changes observed in the RNA-sequencing study. These findings indicate that fat and cholesterol synergize to alter macrophage phenotype, and they also challenge the dogma that KCs are purely proinflammatory in NASH. Conclusion: This comprehensive view of macrophage populations in NASH indicates mechanisms by which cholesterol contributes to NASH progression and identifies potential therapeutic targets for this common disease.
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Colesterol en la Dieta/efectos adversos , Macrófagos del Hígado/metabolismo , Hígado/inmunología , Enfermedad del Hígado Graso no Alcohólico/etiología , Animales , Progresión de la Enfermedad , Hepatitis/etiología , Macrófagos del Hígado/ultraestructura , Metabolismo de los Lípidos , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , TranscriptomaRESUMEN
OBJECTIVE: To investigate differences in clinical features between tobacco smoke-induced and biomass fuel-induced chronic obstructive pulmonary disease (COPD). METHODS: We retrospectively analyzed 206 patients with COPD caused by exposure to tobacco smoke and 81 cases of COPD caused by exposure to biomass fuels who received treatment in our hospital between 2011 March and 2014 March. Difference in general health status, clinical symptoms, the dyspnea score, and comorbidities between the two groups were compared. In addition, pulmonary function, grading, and acute exacerbations were also compared. RESULTS: (1) Difference in general health status: Male and female patients with COPD caused by exposure to tobacco smoke were 83.5 and 16.5%, respectively. Male and female patients with COPD caused by exposure to smoke from biomass fuels were 14.8 and 85.2% (χ2 = 27.2, P < 0.05), respectively. Tobacco smoke-induced COPD was more prevalent in men, and COPD caused by exposure to smoke from biomass fuels was more prevalent in women. After gender adjustment, body mass index (BMI) was lower in women with COPD caused by exposure to smoke from biomass fuels than those by tobacco smoke. There was no statistically significant difference in other indicators, such as age. (2): Difference in clinical symptoms: No statistically significant difference in the modified British Medical Research Counsel (mMRC) Questionnaire, a measure of breathlessness, was observed between the two groups. Dyspnea was more common in COPD patients that was caused by exposure to biomass fuels (38.3%) than by tobacco smoke (11.1%) (χ2 = 17.9, P < 0.05). The comorbidities of allergic diseases (such as allergic rhinitis, bronchial asthma) were more prevalent in COPD patients that was caused by exposure to smoke from biomass fuels (43.2%) than by tobacco smoke (18%) (χ2 = 16.1, P < 0.05). However, COPD comorbid with lung cancer was more prevalent in those cases that were caused by exposure to tobacco smoke (7.77%) than in cases caused by exposure to smoke from biomass fuels (3.7%) (χ2 = 9.7, P < 0.05). (3) Differences in grading of pulmonary function: After gender adjustment, patients with COPD caused by exposure to biomass fuels were mostly in grade B or D. (4) Exacerbations: No significant difference in exacerbations per year was noted between the two groups. CONCLUSIONS: Marked differences exist between patients with COPD caused by exposure to tobacco smoke and smoke from biomass fuels. Patients with COPD caused by exposure to biofuels are mostly females with lower BMI and often with many clinical symptoms and complications, such as allergic rhinitis and bronchial asthma. Such patients are often in stage B or D. Tobacco smoke-induced COPD is more prevalent in male patients, often with complications in the form of lung cancer.
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Nonalcoholic fatty liver disease (NAFLD) affects a large proportion of the American population. The spectrum of disease ranges from bland steatosis without inflammation to nonalcoholic steatohepatitis and cirrhosis. Bile acids are critical regulators of hepatic lipid and glucose metabolism and signal through two major receptor pathways: farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily, and TGR5, a G protein-coupled bile acid receptor (GPBAR1). Both FXR and TGR5 demonstrate pleiotropic functions, including immune modulation. To evaluate the effects of these pathways in NAFLD, we treated obese db/db mice with a dual FXR/TGR5 agonist (INT-767) for 6 weeks. Treatment with the agonist significantly improved the histological features of nonalcoholic steatohepatitis. Furthermore, treatment increased the proportion of intrahepatic monocytes with the anti-inflammatory Ly6C(low) phenotype and increased intrahepatic expression of genes expressed by alternatively activated macrophages, including CD206, Retnla, and Clec7a. In vitro treatment of monocytes with INT-767 led to decreased Ly6C expression and increased IL-10 production through a cAMP-dependent pathway. Our data indicate that FXR/TGR5 activation coordinates the immune phenotype of monocytes and macrophages, both in vitro and in vivo, identifying potential targeting strategies for treatment of NAFLD.
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Hígado Graso/metabolismo , Hígado/metabolismo , Monocitos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Hígado Graso/inmunología , Hígado Graso/patología , Regulación de la Expresión Génica/inmunología , Humanos , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/inmunología , Interleucina-10/biosíntesis , Interleucina-10/inmunología , Lectinas Tipo C/biosíntesis , Lectinas Tipo C/inmunología , Hígado/inmunología , Hígado/patología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/biosíntesis , Lectinas de Unión a Manosa/inmunología , Ratones , Ratones Obesos , Monocitos/inmunología , Monocitos/patología , Enfermedad del Hígado Graso no Alcohólico , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/inmunología , Receptores Citoplasmáticos y Nucleares/inmunología , Receptores Acoplados a Proteínas G/inmunologíaRESUMEN
OBJECTIVE: To investigate the effects and mechanism of pharmacological ascorbate against Influenza A/CA/7/09 (H1N12009). METHODS: NHBE cells (≈ 95% confluent monolayer) in 12-well plates (Corning) were kept at 37°C at all times. NHBE cells were exposed to A/CA/7/09 (H1N12009) influenza virus at MOI of 0.01 for 1 h, rinsed with NHBE medium, and incubated with NHBE medium containing 20 mmol/L ascorbate or 20 mmol/L ascorbate +600 IU/ml Catalase. The cells were then incubated for an additional 4 - 12 h and the culture medium was harvested for titration. Viral titers were determined as log(10) 50% tissue culture infective doses (TCID50) assay in MDCK cells. Ascorbate in NHBE medium was determined using HPLC separation coupled with coulometric electrochemical detection. Hydrogen peroxide was detected indirectly by Clark-type oxygen electrode. RESULTS: In vitro experiments showed that pharmacological ascorbate killed not only isolated viruses, but also viruses from normal human bronchial epithelial cells. The antiviral effect of ascorbic acid appeared to be dose-dependent. 2.5 mmol/L ascorbic acid was able to eliminate 90% of the viruses and 20 mmol/L ascorbic acid totally blocked viral replication in vitro. The antiviral effect of pharmacological ascorbate varied at different phases of infection. Pharmacological ascorbate eliminated viral infectivity with treatment times as short as 4 hours at early stage of infection. But the effect was reversed by catalase. CONCLUSION: Pharmacological ascorbate (vitamin C) as a pro-drug eliminates or kills influenza virus, probable by producing steady-state concentrations of hydrogen peroxide (H2O2) in extracellular fluid.
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Antivirales/farmacología , Ácido Ascórbico/farmacología , Orthomyxoviridae/efectos de los fármacos , Antivirales/administración & dosificación , Ácido Ascórbico/administración & dosificación , Células Cultivadas , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Células Epiteliales/virología , Humanos , Peróxido de Hidrógeno/farmacología , Sistema Respiratorio/citologíaRESUMEN
Glutathione (GSH) is a critical antioxidant for protecting the airway epithelium from oxidant injury and its levels are mainly controlled by glutamate-cysteine ligase (GCL), which is the rate-limiting enzyme in GSH synthesis. A full understanding of the gene regulation mechanism of this important enzyme may disclose the role it plays in respiratory diseases. GCL is made up of two differentially regulated subunits, a catalytic or heavy subunit (GCLC) and a modifier or light subunit (GCLM). Many studies in this field led to the findings of important positive regulatory regions of the GCLC promoter. For a detailed analysis of this gene regulation in the respiratory system, we cloned a 1.76-kb 5'-flanking region of the rat GCLC gene, inserted into a luciferase reporter vector. Exonuclease III was used to cut the 5'-flanking region of the rat GCLC gene unidirectionally into deletion mutants of different lengths. Sequential deletion analysis revealed that regions from -403 to -111 and from -705 to -613 are involved in positive regulation and the region from -745 to -705 is involved in negative regulation of the GCL gene in rat lung epithelial L2 cells. Specific proteins binding to these regions were confirmed by electrophoretic mobility-shift assays (EMSAs) and antibody supershift assays. An E-box motif was found in the negative regulatory region -745 to -705. Site-directed mutagenesis proved that the functional element in this negative regulatory region was a putative E-box element. EMSA and supershift assays showed that USF1 and USF2 can specifically bind to the E-box element. Overexpression of USFs in L2 cells led to a decreased activity of the GCLC promoter. Western blotting demonstrated that the expression of GCLC protein was decreased in the retroviral USFs-expressing cells than in nontransfected (no DNA added) cells, suggesting that USF binding to the E-box at -729/-724 serves to trans-repress GCLC gene expression. These findings indicate that the E-box is an important transcriptional suppressor element in the GCLC promoter in rat lung epithelial L2 cells. Inhibition of interaction between the E-box and the USF may provide an effective means of ameliorating oxidant injury of the lung.
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Elementos E-Box/genética , Regulación Enzimológica de la Expresión Génica , Glutamato-Cisteína Ligasa/genética , Pulmón/enzimología , Factores Estimuladores hacia 5'/metabolismo , Región de Flanqueo 5'/genética , Animales , Anticuerpos/inmunología , Células Cultivadas , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Células Epiteliales/enzimología , Glutamato-Cisteína Ligasa/metabolismo , Pulmón/citología , Regiones Promotoras Genéticas , Ratas , Factores Estimuladores hacia 5'/inmunologíaRESUMEN
OBJECTIVE: To validate that the E-box element in the -745-705 region of rat gamma-GCS catalytic subunit gene (GCLC gene), already found to be a negative regulatory region, is an important transcriptional suppressor element. METHODS: Electrophoretic mobility shift assays (EMSA) and antibody supershift assay were used to confirm the specific binding of USF, a transcription factor that binds the regulatory sequence -745 - 705, and E-box element. Rat alveolar epidermal cells of the line CCL-149 were cultured and then divided into 4 groups: group 1 to be transfected with GCLC-Luc, group 2 to be co-transfected with pGCLC-Luc and pCMV-USF1, group 3 to be co-transfected with pGCLC-Luc and pCMV-USF2, and group 4 to be co-transfected with pGCLC-Luc and blank vector pCMV. Recombinant retrovirus vector PLXSN-USF1 and recombinant retrovirus vector PLXSN-USF2 were constructed. Another CCL-149 cells were divided into 4 groups: group A to be infected with recombinant USF1 virus, group B to be infected with recombinant USF2 virus, group C to be infected with blank vector, and group D not to be infected with any plasmid. Western blotting was used to detect the concentrations of GCLC protein. RESULTS: EMSA showed that only the probes with complete E-box element could be bound by the USFs. Supershift assay showed that the transcription factor binding the probe was USF. The luciferase activity of the CCL-149 cells co-transfected with pCMV-USF1/USF2 and GCLC-Luc were decreased by 66.4% and 63.2% respectively in comparison with those transfected with GCLC-Luc only (both P < 0.05) and decreased by 54.5% and 61.1% respectively in comparison with those of the cells of the blank vector group (both P < 0.05). Western blotting showed that the expression of GCLC protein of the CCL-149 cells transfected with PLXSN-USF1 and PLXSN-USF2 were decreased in comparison with that of the control cells. CONCLUSION: The interaction between E-box and USF suppresses the expression of GCLC gene. E-box is an important transcriptional suppressor element of gamma-GCS gene.
Asunto(s)
Elementos E-Box/genética , Alveolos Pulmonares/citología , gamma-Glutamilciclotransferasa/genética , Animales , Células Cultivadas , Ratas , Elementos de Respuesta , Transcripción Genética , Activación TranscripcionalRESUMEN
OBJECTIVE: To analyze the characteristic of regulatory elements and corresponding transcriptional factors in the 5'-flanking region of rat gamma-glutamylcysteine synthetase (gamma-GCS) catalytic subunit (GCLC) gene. METHODS: A 1 760 bp 5'-flanking region of the rat GCLC was cloned and constructed into pGL-3 enhancer vector which includes luciferase reporter gene. Exonuclease III was used to cut the 5'-flanking region of rat GCLC gene unidirectionally into deletion mutants of different length, GCLC-Luc and its deletion mutants were used to transfect rat alveolar epithelium cells CCL-149, then the regulatory region of the gene was determined by luciferase activity assay of the transfected cells. Analysis of the transcription-factor-binding site was done using Transcription Factor Search software to indicate possible transcriptional factors. Electrophoresis mobility shift assay (EMSA) was used to determine the cis-acting elements and transcriptional factors in these regulatory regions. RESULTS: The experiment cloned the upstream regulatory sequence of rat GCLC gene and its reporter vector GCLC-Luc, as well as 11 deletion mutants of GCLC-Luc. Luciferase activity assay of the cells transfected by GCLC-Luc (-1 758/+2-Luc), mutant 1 (-1 231/+2-Luc), mutant 2 (-1 108/+2-Luc), mutant 3 (-1 087/+2-Luc), mutant 4 (-876/+2-Luc), mutant 5 (-745/+2-Luc), mutant 6 (-705/+2-Luc), mutant 8 (-613/+2-Luc), mutant 9 (-595/+2-Luc), mutant 10 (-403/+2-Luc) and mutant 11(-111/+2-Luc) were (90 012 +/- 2 445), (77 652 +/- 840), (149 927 +/- 4 915), (71 588 +/- 1 108), (99 283 +/- 2 612), (75 443 +/- 1 438), (282 772 +/- 7 046), (96 891 +/- 2 275), (148 917 +/- 5 966), (258 991 +/- 5 015) and (895 +/- 49) U, respectively. EMSA proved that activated protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) can bind to the region of -403 to -111 bp; nuclear factor-1 (NF-1) and CCAAT/enhancer binding protein (C/EBP) can bind to the region of -705 to -613 bp; and upstream stimulatory factor (USF) can bind to the region of -745 to -705 bp. CONCLUSIONS: Two DNA regions -403 to -111 bp and -705 to -613 bp of GCLC gene, which can be bound by transcriptional factors such as NF-1, C/EBP, AP-1, and NF-kappaB on EMSA, are involved in positive gene regulation. A newly identified region -745 to -705 bp of GCLC gene, which can be bound by USF, is involved in negative gene regulation, suggesting that the interaction between E-box and USF can inhibit the expression of gamma-GCS.
Asunto(s)
Región de Flanqueo 5'/genética , Regulación Enzimológica de la Expresión Génica , Glutamato-Cisteína Ligasa/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Animales , Dominio Catalítico/genética , Elementos E-Box/genética , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/biosíntesis , Regiones Promotoras Genéticas , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Ratas , Factores Estimuladores hacia 5'RESUMEN
AIM: To clone and express CML66 cDNA and to prepare rabbit anti-CML66 antibody. METHODS: cDNA isolated from the testis using RT-PCR was cloned into pGEMT. After sequencing, the cDNA was inserted into prokaryotic expression vector pET32b(+). The recombinant vector was transformed into BL-21(DE3) through electroporation. 6xHis-tagged CML66 expression was then induced by IPTG. The protein was purified through Ni(2+) affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified protein was injected into rabbits to prepare polyclonal antibody. RESULTS: The cloned cDNA sequence was identical with that previously reported. The target protein was successfully purified. And rabbit's anti-serum with high titer was obtained. CONCLUSION: We have cloned CML66 successfully, expressed and purified the protein in E.coli.Furthermore,rabbit polyclonal antibody has been obtained.