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Background: The prevalence of food allergy (FA) is increasing. Decreases in the diversity of gut microbiota may contribute to the pathogenesis of FA by regulating IgE production of B cells. Intermittent fasting (IF) is a popular diet with the potential to regulate glucose metabolism, boosting immune memory and optimizing gut microbiota. The potential effect of long-term IF on the prevention and treatment of FA is still unknown. Methods: Two IF protocols (16 h fasting/8 h feeding and 24 h fasting/24 h feeding) were conducted on mice for 56 days, while the control mice were free to intake food (free diet group, FrD). To construct the FA model, all mice were sensitized and intragastrical challenged with ovalbumin (OVA) during the second half of IF (day 28 to day 56). Rectal temperature reduction and diarrhea were recorded to evaluate the symptoms of FA. Levels of serum IgE, IgG1, Th1/Th2 cytokines, mRNA expression of spleen T cell related transcriptional factors, and cytokines were examined. H&E, immunofluorescence, and toluidine blue staining were used to assess the structural changes of ileum villi. The composition and abundance of gut microbiota were analyzed by 16srRNA sequencing in cecum feces. Results: The diarrhea score and rectal temperature reduction were lower in the two fasting groups compared to the FrD groups. Fasting was associated with lower levels of serum OVA-sIgE, OVA-sIgG1, interleukin (IL)-4 and IL-5, and mRNA expression of IL-4, IL-5, and IL-10 in the spleen. While no significant association was observed in interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-6, IL-2 levels. Less mast cell infiltration in ileum was observed in the 16h/8h fasting group compared to the FrD group. ZO-1 expression in the ileum of the two fasting groups was higher in IF mice. The 24h/24h fasting reshaped the gut microbiota, with a higher abundance of Alistipes and Rikenellaceae strains compared to the other groups. Conclusion: In an OVA-induced mice FA model, long-term IF may attenuate FA by reducing Th2 inflammation, maintaining the integrity of the intestinal epithelial barrier, and preventing gut dysbiosis.
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Hipersensibilidade Alimentar , Microbioma Gastrointestinal , Camundongos , Animais , Jejum Intermitente , Modelos Animais de Doenças , Interleucina-5 , Hipersensibilidade Alimentar/etiologia , Citocinas/metabolismo , Imunoglobulina E , Diarreia , RNA MensageiroRESUMO
Clear-cell renal cell carcinoma (ccRCC) is the most prevalent renal malignancy. The pathogenesis of the disease is currently poorly understood, and the prognosis is poor. Therefore, in this study, we focused on exploring and identifying genes and signal transduction pathways that are closely related to ccRCC. Differentially expressed genes (DEGs) were analyzed using the renal cell oncogene expression profiles GSE100666 and GSE68417. DAVID evaluation of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses was used. We constructed a protein-protein interaction (PPI) network of DEGS using Cytoscape software and analyzed the submodules with the CytoHubba plugin. Finally, we performed western blot, immunohistochemistry, and PCR validation by collecting tissues, and also utilized cells for in vitro functional analysis of ceruloplasmin (CP). In total, 202 DEGs (52 upregulated and 150 downregulated genes) were identified. Upregulated DEGs are significantly rich in angiogenesis, cell adhesion, and response to hypoxia, whereas downregulated DEGs are involved in intracellular pH regulation, excretion, coagulation, and chloride transmembrane transport. We selected the interactions of the top 20 hub genes provided by the PPI network, all of which are involved in important physiological pathways in vivo, such as complement and coagulation cascades. Tissue protein assays demonstrated that renal cancer highly expressed CP, while in vitro experiments showed that CP could promote the invasion of renal cancer cells. Our study suggests that ALB, C3, LOX, HRG, CXCR4, GPC3, SLC12A3, CP, and CASR may be involved in the development of ccRCC, and is expected to provide theoretical support for future studies on the diagnosis and targeted therapy of ccRCC.
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Carcinoma de Células Renais/genética , Ceruloplasmina/genética , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Ceruloplasmina/metabolismo , Biologia Computacional/métodos , Bases de Dados Genéticas , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Ontologia Genética , Redes Reguladoras de Genes/genética , Glipicanas , Humanos , Neoplasias Renais/genética , Oncogenes , Prognóstico , Mapas de Interação de Proteínas/genética , Receptores CXCR4 , Membro 3 da Família 12 de Carreador de Soluto , Taxa de SobrevidaRESUMO
BACKGROUND: The coronavirus disease 2019 (COVID-19) has become a global pandemic, with 10%-20% of severe cases and over 508 000 deaths worldwide. OBJECTIVE: This study aims to address the risk factors associated with the severity of COVID-19 patients and the mortality of severe patients. METHODS: 289 hospitalized laboratory-confirmed COVID-19 patients were included in this study. Electronic medical records, including patient demographics, clinical manifestation, comorbidities, laboratory tests results, and radiological materials, were collected and analyzed. According to the severity and outcomes of the patients, they were divided into three groups: nonsurvived (n = 49), survived severe (n = 78), and nonsevere (n = 162) groups. Clinical, laboratory, and radiological data were compared among these groups. Principal component analysis (PCA) was applied to reduce the dimensionality and visualize the patients on a low-dimensional space. Correlations between clinical, radiological, and laboratory parameters were investigated. Univariate and multivariate logistic regression methods were used to determine the risk factors associated with mortality in severe patients. Longitudinal changes of laboratory findings of survived severe cases and nonsurvived cases during hospital stay were also collected. RESULTS: Of the 289 patients, the median age was 57 years (range, 22-88) and 155 (53.4%) patients were male. As of the final follow-up date of this study, 240 (83.0%) patients were discharged from the hospital and 49 (17.0%) patients died. Elder age, underlying comorbidities, and increased laboratory variables, such as leukocyte count, neutrophil count, neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP), procalcitonin (PCT), D-dimer, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) on admission, were found in survived severe cases compared to nonsevere cases. According to the multivariate logistic regression analysis, elder age, a higher number of affected lobes, elevated CRP levels on admission, increased prevalence of chest tightness/dyspnea, and smoking history were independent risk factors for death of severe patients. A trajectory in PCA was observed from "nonsevere" toward "nonsurvived" via "severe and survived" patients. Strong correlations between the age of patients, the affected lobe numbers, and laboratory variables were identified. Dynamic changes of laboratory findings of survived severe cases and nonsurvived cases during hospital stay showed that continuing increase of leukocytes and neutrophil count, sustained lymphopenia and eosinopenia, progressing decrease in platelet count, as well as high levels of NLR, CRP, PCT, AST, BUN, and serum creatinine were associated with in-hospital death. CONCLUSIONS: Survived severe and nonsurvived COVID-19 patients had distinct clinical and laboratory characteristics, which were separated by principle component analysis. Elder age, increased number of affected lobes, higher levels of serum CRP, chest tightness/dyspnea, and smoking history were risk factors for mortality of severe COVID-19 patients. Longitudinal changes of laboratory findings may be helpful in predicting disease progression and clinical outcome of severe patients.
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COVID-19/sangue , COVID-19/mortalidade , COVID-19/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , SARS-CoV-2 , Adulto JovemRESUMO
The pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an unprecedented global social and economic impact, and high numbers of deaths. Many risk factors have been identified in the progression of COVID-19 into a severe and critical stage, including old age, male gender, underlying comorbidities such as hypertension, diabetes, obesity, chronic lung diseases, heart, liver and kidney diseases, tumors, clinically apparent immunodeficiencies, local immunodeficiencies, such as early type I interferon secretion capacity, and pregnancy. Possible complications include acute kidney injury, coagulation disorders, thoromboembolism. The development of lymphopenia and eosinopenia are laboratory indicators of COVID-19. Laboratory parameters to monitor disease progression include lactate dehydrogenase, procalcitonin, high-sensitivity C-reactive protein, proinflammatory cytokines such as interleukin (IL)-6, IL-1ß, Krebs von den Lungen-6 (KL-6), and ferritin. The development of a cytokine storm and extensive chest computed tomography imaging patterns are indicators of a severe disease. In addition, socioeconomic status, diet, lifestyle, geographical differences, ethnicity, exposed viral load, day of initiation of treatment, and quality of health care have been reported to influence individual outcomes. In this review, we highlight the scientific evidence on the risk factors of severity of COVID-19.
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COVID-19 , Estado Terminal , Progressão da Doença , Feminino , Humanos , Masculino , Fatores de Risco , SARS-CoV-2RESUMO
The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a rapidly spreading illness, coronavirus disease 2019 (COVID-19), affecting more than seventeen million people around the world. Diagnosis and treatment guidelines for clinicians caring for patients are needed. In the early stage, we have issued "A rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-nCoV) infected pneumonia (standard version)"; now there are many direct evidences emerged and may change some of previous recommendations and it is ripe for develop an evidence-based guideline. We formed a working group of clinical experts and methodologists. The steering group members proposed 29 questions that are relevant to the management of COVID-19 covering the following areas: chemoprophylaxis, diagnosis, treatments, and discharge management. We searched the literature for direct evidence on the management of COVID-19, and assessed its certainty generated recommendations using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. Recommendations were either strong or weak, or in the form of ungraded consensus-based statement. Finally, we issued 34 statements. Among them, 6 were strong recommendations for, 14 were weak recommendations for, 3 were weak recommendations against and 11 were ungraded consensus-based statement. They covered topics of chemoprophylaxis (including agents and Traditional Chinese Medicine (TCM) agents), diagnosis (including clinical manifestations, reverse transcription-polymerase chain reaction (RT-PCR), respiratory tract specimens, IgM and IgG antibody tests, chest computed tomography, chest x-ray, and CT features of asymptomatic infections), treatments (including lopinavir-ritonavir, umifenovir, favipiravir, interferon, remdesivir, combination of antiviral drugs, hydroxychloroquine/chloroquine, interleukin-6 inhibitors, interleukin-1 inhibitors, glucocorticoid, qingfei paidu decoction, lianhua qingwen granules/capsules, convalescent plasma, lung transplantation, invasive or noninvasive ventilation, and extracorporeal membrane oxygenation (ECMO)), and discharge management (including discharge criteria and management plan in patients whose RT-PCR retesting shows SARS-CoV-2 positive after discharge). We also created two figures of these recommendations for the implementation purpose. We hope these recommendations can help support healthcare workers caring for COVID-19 patients.
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Quimioprevenção/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Adulto , Betacoronavirus , COVID-19 , Teste para COVID-19 , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/prevenção & controle , Medicina Baseada em Evidências , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias/prevenção & controle , Alta do Paciente/normas , Pneumonia Viral/diagnóstico , Pneumonia Viral/prevenção & controle , Guias de Prática Clínica como Assunto , SARS-CoV-2RESUMO
BACKGROUND: ß2 receptor agonists induce airway smooth muscle relaxation by increasing intracellular cAMP production. PKA is the traditional downstream signaling pathway of cAMP. Exchange protein directly activated by cAMP (Epac) was identified as another important signaling molecule of cAMP recently. The role of Epac in asthmatic airway inflammation and airway remodeling is unclear. METHODS: We established OVA-sensitized and -challenged acute and chronic asthma mice models to explore the expression of Epac at first. Then, airway inflammation and airway hyperresponsiveness in acute asthma mice model and airway remodeling in chronic asthma mice model were observed respectively after treatment with Epac-selective cAMP analogue 8-pCPT-2'-O-Me-cAMP (8pCPT) and Epac inhibitor ESI-09. Next, the effects of 8pCPT and ESI-09 on the proliferation and apoptosis of in vitro cultured mouse airway smooth muscle cells (ASMCs) were detected with CCK-8 assays and Annexin-V staining. Lastly, the effects of 8pCPT and ESI-09 on store-operated Ca2+ entry (SOCE) of ASMCs were examined by confocal Ca2+ fluorescence measurement. RESULTS: We found that in lung tissues of acute and chronic asthma mice models, both mRNA and protein expression of Epac1 and Epac2, two isoforms of Epac, were lower than that of control mice. In acute asthma mice model, the airway inflammatory cell infiltration, Th2 cytokines secretion and airway hyperresponsiveness were significantly attenuated by 8pCPT and aggravated by ESI-09. In chronic asthma mice model, 8pCPT decreased airway inflammatory cell infiltration and airway remodeling indexes such as collagen deposition and airway smooth muscle cell proliferation, while ESI-09 increased airway inflammation and airway remodeling. In vitro cultured mice ASMCs, 8pCPT dose-dependently inhibited, whereas ESI-09 promoted ASMCs proliferation. Interestingly, 8pCPT promoted the apoptosis of ASMCs, whereas ESI-09 had no effect on ASMCs apoptosis. Lastly, confocal Ca2+ fluorescence examination found that 8pCPT could inhibit SOCE in ASMCs at 100 µM, and ESI-09 promoted SOCE of ASMCs at 10 µM and 100 µM. In addition, the promoting effect of ESI-09 on ASMCs proliferation was inhibited by store-operated Ca2+ channel blocker, SKF-96365. CONCLUSIONS: Our results suggest that Epac has a protecting effect on asthmatic airway inflammation and airway remodeling, and Epac reduces ASMCs proliferation by inhibiting SOCE in part.
Assuntos
Remodelação das Vias Aéreas , Asma/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Pulmão/metabolismo , Pneumonia/metabolismo , Hipersensibilidade Respiratória/metabolismo , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Apoptose , Asma/induzido quimicamente , Asma/tratamento farmacológico , Asma/fisiopatologia , Sinalização do Cálcio , Proliferação de Células , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Modelos Animais de Doenças , Feminino , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Hidrazonas/farmacologia , Mediadores da Inflamação/metabolismo , Isoxazóis/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos Endogâmicos BALB C , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Ovalbumina , Pneumonia/induzido quimicamente , Pneumonia/fisiopatologia , Pneumonia/prevenção & controle , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/tratamento farmacológico , Hipersensibilidade Respiratória/fisiopatologiaRESUMO
OBJECTIVE: The accurate diagnosis of undetermined pancreaticobiliary strictures remains challenging. Current ERCP-guided tissue sampling methods are of low sensitivity. Confocal laser endomicroscopy (CLE) is a new procedure and allows real optical biopsies that may improve the diagnosis of undetermined pancreaticobiliary strictures. The aim of this meta-analysis was to determine the diagnostic yield of CLE, tissue sampling, and CLE combined with tissue sampling for undetermined pancreaticobiliary strictures. METHOD: Pubmed, Embase, and the Cochrane Library database were reviewed for relevant studies. Pooled estimates of sensitivity and specificity with 95% confidence intervals (CIs) were calculated using the random-effects meta-analysis model. The summary receiver-operating characteristic (SROC) curve was constructed, and the area under the receiver operating characteristic curve (AUC) was calculated. RESULTS: Twelve studies involving 591 patients were enrolled in our analysis. The overall sensitivity and the specificity estimate of CLE for discriminating benign and malignant pancreaticobiliary strictures were 87% (95%CI, 83-91%) and 76% (95%CI, 70-81%), respectively. The AUC to assess the diagnostic efficacy was 0.8705. For tissue sampling, the overall sensitivity and the specificity estimate were 64% (95%CI, 57-70%) and 94% (95%CI, 90-97%), respectively. The AUC to assess the diagnostic efficacy was 0.8040. A combination of both methods increased the sensitivity (93%; 95%CI, 88-96%) with a specificity of 82% (95%CI, 74-89%). The AUC to assess the diagnostic efficacy was 0.9377. There was no publication bias by Deeks' Funnel Plot with p = .936. CONCLUSIONS: Compared with tissue sampling, CLE may increase the sensitivity for the diagnosis of malignant pancreaticobiliary strictures. A combination of both can effectively diagnose malignant pancreaticobiliary strictures.
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Biópsia/métodos , Constrição Patológica/diagnóstico , Endoscopia Gastrointestinal , Microscopia Confocal/métodos , Constrição Patológica/etiologia , Humanos , Curva ROCRESUMO
BACKGROUND: Circulating fibrocytes (CFs) have been shown to participate in subepithelial fibrosis of asthma with chronic airflow limitation by acting as an important source of fibroblasts deposited beneath airway epithelia. Serum amyloid P (SAP) is an innate inhibitor of fibrocytes differentiation. Store-operated Ca2+ entry (SOCE) is the major Ca2+ influx of non-excitable cells. In this study, the role of SOCE in the regulation of fibrocytes differentiation and the effects of Th2 cytokine IL-4 and SAP on SOCE of fibrocytes were investigated. METHODS: Peripheral blood mononuclear cells or monocytes were cultured in serum-free medium for 7days to differentiate into fibrocytes; the expression of SOC channels was determined with PCR, SOCE was measured with Ca2+ fluorescence imaging. RESULTS: IL-4 significantly promoted monocyte derived fibrocytes differentiation in vitro. It also increased both SOCE which was induced by thapsigargin or UTP and molecules STIM1 and Orai1 which were related to expression of SOC channels in fibrocytes. Fibrocytes differentiation induced by IL-4 and SOC channels activity could be inhibited by SOC channel blocker SKF-96365. As expected, SAP significantly inhibited IL-4-induced differentiation of fibrocytes, the activity of SOCE and the expression of STIM1 and Orai1 in IL-4-treated fibrocytes. CONCLUSION: IL-4 and SAP reversely regulates cultured fibrocytes differentiation in vitro by respectively promoting or inhibiting the expression and activity of SOC channels in fibrocytes.
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Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Interleucina-4/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas de Neoplasias/efeitos dos fármacos , Proteína ORAI1/efeitos dos fármacos , Componente Amiloide P Sérico/farmacologia , Molécula 1 de Interação Estromal/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Imidazóis/farmacologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Fatores de TempoRESUMO
Persistent air leak (PAL) is associated with significant morbidity and mortality, prolonged hospitalization and increased health-care costs. It can arise from a number of conditions, including pneumothorax, necrotizing infection, trauma, malignancies, procedural interventions and complications after thoracic surgery. Numerous therapeutic options, including noninvasive and invasive techniques, are available to treat PALs. Recently, endobronchial one-way valves have been used to treat PAL. We conducted a systematic review based on studies retrieved from PubMed, EMbase and Cochrane library. We also did a hand-search in the bibliographies of relevant articles for additional studies. 34 case reports and 10 case series comprising 208 patients were included in our review. Only 4 patients were children, most of the patients were males. The most common underlying disease was COPD, emphysema and cancer. The most remarkable cause was pneumothorax. The upper lobes were the most frequent locations of air leaks. Complete resolution was gained within less than 24 h in majority of patients. Complications were migration or expectoration of valves, moderate oxygen desaturation and infection of related lung. No death related to endobronchial one-way valves implantation has been found. The use of endobronchial one-way valve adds to the armamentarium for non-invasive treatments of challenging PAL, especially those with difficulties of anesthesia, poor condition and high morbidity. Nevertheless, prospective randomized control trials with large sample should be needed to further evaluate the effects and safety of endobronchial one-way valve implantation in the treatment of PAL.
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Broncoscopia/métodos , Endoscopia/métodos , Pneumopatias/cirurgia , Complicações Pós-Operatórias/cirurgia , Humanos , Pneumopatias/diagnóstico por imagem , Pneumopatias/etiologia , Pneumotórax/diagnóstico por imagem , Pneumotórax/etiologia , Pneumotórax/cirurgia , Complicações Pós-Operatórias/diagnóstico por imagem , Complicações Pós-Operatórias/etiologiaRESUMO
OBJECTIVE: We retrospectively reviewed the accuracy of conventional transbronchial needle aspiration (cTBNA) in the subtyping of lesions located in or around central airways by comparing the histological diagnosis based on TBNA and surgical specimens. MATERIALS AND METHODS: cTBNA was conducted in consecutive patients with lesions located in or around the central airways (trachea, left and right primary bronchi, hilar and mediastinal masses or lymph nodes) between October 2012 and May 2014 in Wuhan No. 1 Hospital. The aspirated specimens in all patients were performed cytological and/or histopathological examination. Of these patients, some were subjected to surgical resection and histopathological examination was performed by the Department of Pathology. In the patients with gross specimens, the final diagnosis was established based on histopathological results from these specimens. RESULTS: In 63 patients diagnosed with cTBNA for the lesions located in or around the central airways, 23 patients with a diagnosis of lung cancer or atypical hyperplasia underwent surgery. The final diagnosis based on histopathology of surgery specimen was lung cancer in 22 patients [3 small cell lung cancer (SCLC), 9 squamous cell carcinoma (SCC), 5 adenocarcinoma (ADC), 4 adenosquamous carcinoma (ADS) and 1 neuroendocrine carcinoma], and inflammatory pseudotumor in 1 patient. The overall diagnostic yield of cTBNA for lung cancer was 95.7% (22/23), but the accuracy for histological typing of lung cancer is only 63.6% (14/22), for adenosquamous lung carcinoma was only 25% (1/4). CONCLUSIONS: cTBNA is a safe and effective procedure that can be used for the diagnosis of central lung cancer. However, the accuracy of TBNA for the histological classification of lung cancer is relatively low, especially for adenosquamous lung carcinoma.
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OBJECTIVE: Mast cell infiltration into airway smooth muscle (ASM) bundle is an important feature of asthma. Extracellular adenosine triphosphate (eATP) contributes to the initiation of airway inflammation. eATP induces mast cells migration by acting through purinergic receptors. CD39 is an ectonucleotidase that degrades ATP to ADP and AMP. Whether eATP participates in the migration of mast cell towards ASM cells is still unknown. METHODS: Airway smooth muscle cells (ASMCs) were isolated from C57/BL6J mice sensitized and challenged with OVA. ASMCs were in vitro cultured and stimulated with IL-4 + IL-13 in the presence or absence of exogenous CD39 or CD39 inhibitor ARL67156. ATP level in the supernatants was measured with ATP content determination kit. CXCL10 concentration in the ASMCs supernatants was measured by ELISA, the mRNA expression of CXCL10 in ASMCs was determined with real-time PCR. Human mast cell line HMC-1 was cultured in Iscove's-Modified Dubecco's Medium. The expression of CXCR3 in HMC-1 cells was determined with flow cytometry and real-time PCR, respectively. HMC-1 migration rates were determined with transwell system. RESULTS: In the supernatants of Th2 cytokine-stimulated ASMCs, ATP level was higher than that without stimulation. CD39 decreased, whereas ARL67156 increased ATP level in the supernatants. Both ATP and the supernatants of Th2 cytokine-stimulated ASMCs induced migration of HMC-1 cells. The surface and mRNA expression of CXCR3 in HMC-1 cells, and the mRNA expression and secretion of CXCL10 in ASMCs were increased after stimulation with ATP or Th2 cytokines. All these effects were partially inhibited by CD39. CONCLUSION: Our data suggested ASMCs in the asthma microenvironment promoted the migration of mast cells via secretion of ATP and the expression of CXCL10/CXCR3 axis. CD39 could reverse this effect and may be a new target for the treatment of asthma.
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Trifosfato de Adenosina/imunologia , Antígenos CD/imunologia , Apirase/imunologia , Asma/imunologia , Quimiotaxia/imunologia , Mastócitos/imunologia , Músculo Liso/imunologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Apirase/antagonistas & inibidores , Linhagem Celular , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/imunologia , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores CXCR3/genética , Receptores CXCR3/imunologiaRESUMO
Plasma membrane Ca2+-ATPase (PMCA) plays an important role in regulating intracellular Ca2+ homeostasis by extruding excessive Ca2+ to extracellular spaces. PMCA has four isoforms and is widely expressed in different tissues and cells including airway smooth muscle cells (ASMCs). In the present study, we investigated the role of PMCA in the maintenance of Ca2+ homeostasis and regulation of ASMCs proliferation. By using Ca2+ fluorescence, we found that inhibition of PMCA with LaCl3 or carboxyeosin (CE) decreased the decay rate of Ca2+ transient induced by bradykinin (BK). No obvious decay was observed when SERCA was inhibited by thapsigargin (TpG). LaCl3 and CE also induced a spontaneous [Ca2+]i increase in the presence of TpG even in Ca2+-free bath solution. Both LaCl3 and CE inhibited UTP-induced Ca2+ oscillations in ASMCs. PCR assay found that PMCA1 and PMCA4 mRNA were expressed in rat ASMCs. The expression of PMCA4 was downregulated in proliferating ASMCs when compared to resting cells. Both the isoform-nonselective PMCA inhibitor caloxin 2a1 and PMCA4-selective inhibitor caloxin 1b1 decreased the decay rate of Ca2+ transient induced by TpG or BK. PMCA inhibitors except caloxin 2a1 promoted ASMCs proliferation. Annexin-V apoptosis assay detected that caloxin 2a1 increased ASMCs apoptosis, suggesting that inhibition of PMCA with different blockers results in different [Ca2+]i and thus different cellular response. Our results provide evidences to support the hypothesis that PMCA is involved in the regulation of Ca2+ homeostasis and ASMCs proliferation. These data suggest that PMCA may be a new target in the treatment of chronic asthma.
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Sinalização do Cálcio , Miócitos de Músculo Liso/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Brônquios/citologia , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular , Lantânio/farmacologia , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Peptídeos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
Store-operated calcium entry (SOCE) is the main Ca(2+) influx pathway of dendritic cells (DCs). DCs primed with histamine facilitate Th2 immune response via different types of histamine receptors. Histamine induces DCs to release Ca(2+) from internal store. Therefore, we wonder that whether histamine could activate SOCE in DCs through its receptors, and what's the functional relevance of the Ca(2+) influx through SOCE induced by histamine in Th(2) response. We certificate that histamine induced a transient Ca(2+) release followed by pronounced Ca(2+) influx after re-addition of external Ca(2+) which could be inhibited by SOCE blockers SKF-96365 and BTP-2. Moreover, the percentages of DCs that showed an obvious Ca(2+) release response to histamine were decreased in the presence of histamine 1 (H1) receptor antagonist pyridylethylamine (Pyr) or histamine 4 (H4) receptor antagonist JNJ7777120 (JNJ). Histamine up-regulated the mRNA expression of STIM1 in DCs, one of the two major proteins of SOCE channel. SOCE blocker BTP-2 and histamine receptor antagonists JNJ and Pyr inhibited the increase of CD86 induced by histamine on DCs. Histamine increased the level of IL-10 and decreased the level of IL-12p70 secreted by DCs. SOC blockers SKF and BTP-2 inhibited the level of both IL-10 and IL-12p70 secreted by DCs. Pretreatment of SOC blockers and H1, H4 receptor antagonists with DCs inhibited the Th2 polarization of T helper cells induced by histamine in mixed lymphocyte responses (MLR). We demonstrated that SOCE was involved in histamine-induced maturation and Th(2) response of DCs which was through histamine 1 and 4 receptor.
Assuntos
Cálcio/metabolismo , Células Dendríticas/metabolismo , Histamina/farmacologia , Monócitos/metabolismo , Células Th2/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células do Cúmulo , Células Dendríticas/efeitos dos fármacos , Histamina/metabolismo , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Monócitos/efeitos dos fármacos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/metabolismo , Molécula 1 de Interação Estromal , Células Th2/efeitos dos fármacosRESUMO
STIM1 'senses' decreases in endoplasmic reticular (ER) luminal Ca(2+) and induces store-operated Ca(2+) (SOC) entry through plasma membrane Orai channels. The Ca(2+)/calmodulin-activated K(+) channel K(Ca)3.1 (previously known as SK4) has been implicated as an 'amplifier' of the Ca(2+)-release activated Ca(2+) (CRAC) current, especially in T lymphocytes. We have previously shown that human macrophages express K(Ca)3.1, and here we used the whole-cell patch-clamp technique to investigate the activity of these channels during Ca(2+) store depletion and store-operated Ca(2+) influx. Using RT-PCR, we found that macrophages express the elementary CRAC channel components Orai1 and STIM1, as well as Orai2, Orai3 and STIM2, but not the putatively STIM1-activated channels TRPC1, TRPC3-7 or TRPV6. In whole-cell configuration, a robust Ca(2+)-induced outwardly rectifying K(+) current inhibited by clotrimazole and augmented by DC-EBIO could be detected, consistent with K(Ca)3.1 channel current (also known as intermediate-conductance IK1). Introduction of extracellular Ca(2+) following Ca(2+) store depletion via P2Y(2) receptors induced a robust charybdotoxin (CTX)- and 2-APB-sensitive outward K(+) current and hyperpolarization. We also found that SOC entry induced by thapsigargin treatment induced CTX-sensitive K(+) current in HEK293 cells transiently expressing K(Ca)3.1. Our data suggest that SOC and K(Ca)3.1 channels are tightly coupled, such that a small Ca(2+) influx current induces a much large K(Ca)3.1 channel current and hyperpolarization, providing the necessary electrochemical driving force for prolonged Ca(2+) signaling and store repletion.
Assuntos
Canais de Cálcio/biossíntese , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Macrófagos/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Moléculas de Adesão Celular/biossíntese , Charibdotoxina/farmacologia , Clotrimazol/farmacologia , Células HEK293 , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Proteína ORAI1 , Proteína ORAI2 , Técnicas de Patch-Clamp , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Uridina Trifosfato/farmacologiaRESUMO
Th2 cytokine interleukin (IL)-13 plays a central role in the pathogenesis of allergic asthma. IL-13 exhibits a direct effect on airway smooth muscle cells (ASMCs) to cause airway hyperresponsiveness. IL-13 has been demonstrated to regulate Ca(2+) signaling in ASMCs, but the underlying mechanisms are not fully understood. Store-operated Ca(2+) entry (SOCE) plays an important role in regulating Ca(2+) signaling and cellular responses of ASMCs, whether IL-13 affects SOCE in ASMCs has not been reported. In this study, by using confocal Ca(2+) fluorescence imaging, we found that IL-13 (10 ng/ml) treatment increased basal intracellular Ca(2+) ([Ca(2+)](i)) level, Ca(2+) release and SOCE induced by SERCA inhibitor thapsigargin in rat bronchial smooth muscle cells. The glucocorticoid dexamethasone and the short-acting beta2 adrenergic agonist (beta2 agonist) salbutamol suppressed IL-13-augumented basal [Ca(2+)](i), Ca(2+) release and SOCE, whereas the long-acting beta2 agonist salmeterol had no effect on altered Ca(2+) signaling in IL-13-treated ASMCs. Membrane-permeable cAMP analog dibutyryl-cAMP (db-cAMP) similarly decreased Ca(2+) release and SOCE induced by thapsigargin in IL-13-treated ASMCs, confirmed a role of cAMP/PKA signaling pathway in the regulation of SOCE. IL-13 promoted the proliferation of ASMCs stimulated by serum; this effect was inhibited by nonspecific Ca(2+) channel blockers SKF-96365 and NiCl(2), by salmeterol, but not by salbutamol and dexamethasone. IL-13 treatment did not change the expression of SOC channel-associated molecules STIM1, Orai1 and TRPC1 at mRNA level. Our findings identified a promoting effect of IL-13 on Ca(2+) release and SOCE in ASMCs, which partially contributes to its effect on the proliferation of ASMCs; the differences of glucocorticoids and beta2 agonists in inhibiting Ca(2+) signal and proliferation potentiated by IL-13 suggest that these therapies of asthma may have distinct effect on the relief of airway contraction and remodeling in bronchial asthma.