The Monocyte-Derived Exosomal CLMAT3 Activates the CtBP2-p300-NF-κB Transcriptional Complex to Induce Proinflammatory Cytokines in ALI.

Monocytes and macrophages are the two major cell types involved in innate immunity. Exosomes act as signaling molecules to regulate cell-to-cell communication by releasing proteins, mRNAs, microRNAs (miRNAs), and long noncoding RNAs (lncRNAs). However, it is still unclear whether monocyte-derived exosomes are involved in the communication between monocytes and macrophages. In this study, we analyzed the differentially expressed lncRNA profiles in monocytes isolated from blood samples of healthy controls and acute lung injury (ALI) patients. We focused our study on investigating the signaling downstream of CLMAT3 (colorectal liver metastasis-associated transcript 3), a lncRNA that regulated proinflammatory cytokine genes. We revealed that CLMAT3 specifically targeted CtBP2 (C-terminal binding protein 2) and repressed its expression. Elevated CtBP2 acted as a coactivator to assemble a transcriptional complex with histone acetyltransferase p300 and NF-κB (nuclear factor κB) subunits. In vitro coculture and in vivo injection of ALI monocyte-derived exosomes increased the production of proinflammatory cytokines. Importantly, the administration of two CtBP2 inhibitors, NSC95397 and MTOB, could significantly reverse CtBP2-mediated transactivation. Collectively, our results support a model in which monocyte-derived exosomal CLMAT3 activates the CtBP2-p300-NF-κB complex to induce proinflammatory cytokines, thus contributing to the pathogenesis of ALI.

Assessing risk factors for SARS-CoV-2 infection in patients presenting with symptoms in Shanghai, China: a multicentre, observational cohort study.

Background: The outbreak of COVID-19 has led to international concern. We aimed to establish an effective screening strategy in Shanghai, China, to aid early identification of patients with COVID-19. Methods: We did a multicentre, observational cohort study in fever clinics of 25 hospitals in 16 districts of Shanghai. All patients visiting the clinics within the study period were included. A strategy for COVID-19 screening was presented and then suspected cases were monitored and analysed until they were confirmed as cases or excluded. Logistic regression was used to determine the risk factors of COVID-19. Findings: We enrolled patients visiting fever clinics from Jan 17 to Feb 16, 2020. Among 53 617 patients visiting fever clinics, 1004 (1·9%) were considered as suspected cases, with 188 (0·4% of all patients, 18·7% of suspected cases) eventually diagnosed as confirmed cases. 154 patients with missing data were excluded from the analysis. Exposure history (odds ratio [OR] 4·16, 95% CI 2·74-6·33; p<0·0001), fatigue (OR 1·56, 1·01-2·41; p=0·043), white blood cell count less than 4 × 109 per L (OR 2·44, 1·28-4·64; p=0·0066), lymphocyte count less than 0·8 × 109 per L (OR 1·82, 1·00-3·31; p=0·049), ground glass opacity (OR 1·95, 1·32-2·89; p=0·0009), and having both lungs affected (OR 1·54, 1·04-2·28; p=0·032) were independent risk factors for confirmed COVID-19. Interpretation: The screening strategy was effective for confirming or excluding COVID-19 during the spread of this contagious disease. Relevant independent risk factors identified in this study might be helpful for early recognition of the disease. Funding: National Natural Science Foundation of China.

Downregulation of miR-199a-3p mediated by the CtBP2-HDAC1-FOXP3 transcriptional complex contributes to acute lung injury by targeting NLRP1.

Emerging evidence indicates that microRNAs (miRNAs) play fundamental roles in the pathogenesis of multiple diseases, including acute lung injury (ALI). Here, we discovered that miR-199a-3p was significantly downregulated in ALI lung tissues using a microarray analysis. In vitro lipopolysaccharide (LPS) treatment of the human epithelial cell line A549 and the human macrophage cell line U937 caused a decrease of miR-199a-3p. Mechanically, miR-199a-3p specifically bound to the 3'-untranslated region (3'-UTR) of NLRP1 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 1), a critical member of inflammasomes. Ectopic overexpression or downregulation of miR-199a-3p resulted in the repression or induction of NLRP1, respectively, thereby downregulating or activating its downstream events. Moreover, transcription factor FOXP3 (forkhead box P3) was able to specifically bind to the promoter of miR-199a-3p. Knockdown or overexpression of FOXP3 resulted in a decrease or induction miR-199a-3p expression, respectively. Using immunoprecipitation (IP), mass spectrometry and co-IP assays, we found that FOXP3 formed a transcriptional complex with HDAC1 (histone deacetylase 1) and CtBP2 (C-terminal-binding protein 2). Collectively, our results suggested that the CtBP2-HDAC1-FOXP3 transcriptional complex (CHFTC) could specifically bind to the promoter of miR-199a-3p and repress its expression. Downregulation of miR-199a-3p eliminated its inhibition of NLRP1, causing activation of NLRP1 and cleavage of pro-IL-1ß and pro-IL-18 mediated by Caspase-1. The secretion of IL-1ß and IL-18 further aggravated the inflammatory response and resulted in the occurrence of ALI.

Refractory chronic cough due to gastroesophageal reflux: Definition, mechanism and management.

Refractory chronic cough due to gastroesophageal reflux is a troublesome condition unresponsive to the standard medical anti-reflux therapy. Its underlying mechanisms may include incomplete acid suppression, non-acid reflux, transient lower esophageal sphincter relaxations and esophageal hypersensitivity. The diagnosis of this disorder depends on both the findings of multi-channel intraluminal impedance-pH monitoring and the subsequent intensified anti-reflux therapy. The strategies of pharmacological treatment for refractory chronic cough due to reflux include the optimization of proton pump inhibitors and add-on therapies with histamine H2 receptor antagonists, baclofen and gabapentin. However, the further study is needed to satisfy its management.

Gastro-esophageal reflux induced cough with airway hyperresponsiveness.

To investigate the characteristics of gastroesophageal reflux induced cough (GERC) with airway hyperresponsiveness (AHR). Compared to patients with GERC alone and healthy subjects, cough sensitivity, multi-channel intraluminal impedance combined with pH monitoring and airway inflammation were evaluated in patients with GERC and AHR. 23 patients were definitely diagnosed as acid reflux induced GERC, 9 patients developed AHR concomitantly. When compared with GERC patients, patients with AHR had significantly increased number of proximal extent episodes (21.5 (28.6) vs. 7.5 (1.8), Z = -2.038, P = 0.042) and increased proportion of proximal extent episodes to total refluxes episodes (24.5 (13.5)% vs. 4.2 (7.3)%, Z = -2.138, P = 0.032), and the level of IL-8 in the airway of these patients was significantly higher than that in healthy subjects (71.1 (64.0) vs. 24.3 (35.2) pg/ml, Z = -2.013, P = 0.044). Gastroesophageal reflux may cause neutrophilic airway inflammation due to the acid reflux into the airway, which results in AHR. However, AHR is not definitely able to cause chronic cough. Thus differential diagnosis is required in clinical practice.

Therapeutic efficacy of baclofen in refractory gastroesophageal reflux-induced chronic cough.

AIM: To evaluate the efficacy and safety of baclofen for treatment of refractory gastroesophageal reflux-induced chronic cough (GERC) unresponsive to standard anti-reflux therapy. METHODS: Sixteen patients with refractory GERC were given an 8-wk course of baclofen 20 mg three times a day as an add-on therapy to omeprazole. Changes in the cough symptom score, cough threshold to capsaicin, reflux symptom score and possible adverse effects were determined after treatment. The variables of multi-channel intraluminal impedance combined with pH monitoring were compared between responders and non-responders to baclofen. RESULTS: Twelve of 16 patients completed treatment. Cough disappeared or improved in 56.3% (9/16) of patients, including 6 patients with acid reflux-induced cough (66.7%) and 3 patients with non-acid reflux-induced cough (33.3%). With baclofen treatment, the cough symptom score began to decrease at week 2, was clearly decreased at week 6 and reached a minimum at week 8. At the end of therapy, the lowest concentration of capsaicin required for induction of ≥ 2 and ≥ 5 coughs increased from 0.98 (1.46) to 1.95 (6.82) µmol/L (Z = -2.281, P = 0.024) and from 1.95 (7.31) to 7.8 (13.65) µmol/L (Z = -2.433, P = 0.014), respectively, and the reflux symptom score decreased from 8.0 ± 1.6 to 6.8 ± 0.8 (t = 2.454, P = 0.023). The number of acid reflux episodes was significantly lower in responders than in non-responders. The main adverse effects were somnolence, dizziness and fatigue. CONCLUSION: Baclofen is a useful, but suboptimal treatment option for refractory GERC.

[Optimal cut-off point of symptom association probability in the diagnosis of gastroesophageal reflux-induced chronic cough].

OBJECTIVE: To explore the optimal cut-off point of symptom association probability (SAP) in the diagnosis of gastroesophageal reflux-induced chronic cough (GERC) and therefore to improve the diagnostic accuracy. METHODS: Patients with suspected GERC consecutively referred to our respiratory clinic were enrolled into this prospective study between July 2011 and February 2013. After multi-channel intraluminal esophageal impedance and pH monitoring, SAP was calculated by associating the cough recordings on the patients' diary with the detected reflux. GERC was confirmed when there was a favorable response to the following anti-reflux therapy despite the laboratory findings. The optimal cutoff point of SAP was defined according to the highest Youden index. Then, the sensitivity, specificity, positive and negative predictive values, the area under the curve of ROC, and the Kappa value for the optimal cut-off point of SAP was calculated and compared to those of SAP standards currently used in China or generally accepted in the diagnosis of GERC. RESULTS: During the study period, 103 patients with suspected GERC were recruited. Among them, GERC was confirmed in 87 patients (84.5%), including 54 patients (62.1%) due to acid reflux and 33 patients (37.9%) due to non-acid reflux. The optimal cut-off point of SAP was defined at ≥ 80% based on the highest Youden index of 0.372. For the diagnosis of GERC, SAP ≥ 80% had the area under the curve of ROC of 0.686, the Kappa value of 0.264, the sensitivity of 74.7%, the specificity of 62.5%, positive predictive value of 91.5% and negative predictive value of 31.3% respectively, which were superior to those of SAP ≥ 75% currently used in China, and to those of SAP ≥ 95% ( the generally accepted cut-off) in that the balance between higher sensitivity and higher specificity was maintained. When combined with DeMeester score ≥ 12.7, the diagnostic accuracy of SAP ≥ 80% was further improved, with the area under the curve of ROC of 0.820, the Kappa value of 0.689, the sensitivity of 87.0%, the specificity of 76.0%, positive predictive value of 94.1% and negative predictive value of 80.0%. CONCLUSION: SAP ≥ 80% may be a more suitable standard for the diagnosis of GERC.

[The diagnostic value of multichannel intraluminal esophageal impedance and pH monitoring in gastroesophageal reflux-related cough].

OBJECTIVE: To evaluate the diagnostic value and limitation of multichannel intraluminal esophageal impedance and pH (MII-pH) monitoring on the diagnosis of gastroesophageal reflux-related chronic cough (GERC). METHODS: The patients with suspicious GERC consecutively referred to our respiratory clinic between May 2010 and July 2011 underwent a MII-pH monitoring, and received anti-reflux drug therapy, irrespective of the laboratory findings. Chronic cough due to gastroesophageal reflux was determined when there was a favorable response to anti-reflux therapy. Then, the sensitivity, specificity, false positive and negative rate, total consistence, positively and negatively predictive value, the area under the curve of ROC and the Kappa value of the laboratory investigation were calculated for the diagnosis of GERC. RESULTS: During the research period, 56 patients completed MII-pH monitoring. Among them, the abnormal reflux was found in 35 patients, and GERC was finally confirmed in 30 patients (85.7%) including 25 patients (83.3%) due to acid reflux and 5 patients (16.7%) due to non-acid reflux. In the remaining 21 patients with normal reflux episodes, 6 patients (28.6%) could be explained by non-acid reflux for their cough because of a relatively predominant weakly acid reflux and favorable response to empirical anti-reflux therapy. For the diagnosis of GERC, MII-pH monitoring had the sensitivity of 83.3%, the specificity of 75.0%, false positive rate of 25.0%, false negative rate of 16.7%, total consistence of 80.4%, positive predictive value of 85.7%, negative predictive value of 71.4%, the area under the curve of ROC of 0.792 and Kappa value of 0.577 respectively. CONCLUSION: MII-pH is a sensitive and reliable tool for the diagnosis of GERC due to its ability to detect both acid and non-acid reflux.

Discrepancy between presumptive and definite causes of chronic cough.

BACKGROUND: The current diagnostic algorithms for chronic cough require the establishment of the primary presumptive causes followed by the confirmation of diagnosis with the specific therapies. The aim of the study was to investigate the discrepancy between presumptive and definite causes and its clinical implication. METHODS: A total of 109 patients with chronic cough underwent laboratory investigations to identify the cause of cough; including sinus computerized tomography (if needed), histamine bronchial provocation, induced sputum cytology and 24-hour esophageal pH or multi-channel intraluminal impedance combined with pH monitoring. The presumptive causes were confirmed by treating them sequentially. The difference between presumptive and definite causes of chronic cough was compared. RESULTS: Single cause was more frequent in the definite diagnosis than in the presumptive diagnosis (78.9% vs. 54.1%, χ(2) = 15.01, P = 0.0001). In contrast, multiple causes were significantly fewer in definite diagnosis than in the presumptive diagnosis (15.6% vs. 37.6%, χ(2) = 13.53, P = 0.0002). There was a discrepancy between definite and presumptive causes in 30 patients (27.5%). Compared with the presumptive causes, definite upper airway cough syndrome (24.8% vs. 11.9%, χ(2) = 6.0, P = 0.01) and gastroesophageal reflux disease (6.4% vs. 0, χ(2) = 7.23, P = 0.007) was more frequent as a single cause of chronic cough while cough variant asthma plus gastroesophageal reflux disease (3.7% vs. 11.9%, χ(2) = 5.17, P = 0.02) and upper airway cough syndrome plus nonasthmatic eosinophilic bronchitis (0 vs. 9.2%, χ(2) = 10.48, P = 0.001) were fewer as multiple causes of chronic cough. CONCLUSIONS: A discrepancy was common between presumptive and definite causes of chronic cough. To treat presumptive causes sequentially may be a suitable solution for avoidance of erroneous multiple causes and possible over-treatment.

[Changes in the spectrum and frequency of causes for chronic cough: a retrospective analysis].

OBJECTIVE: To investigate the changing patterns of the spectrum and frequency of causes for chronic cough, and to explore its clinical implications. METHODS: Nine hundred and forty patients consecutively referred to Department of Respiratory Medicine for evaluation of chronic cough between January 2004 and December 2008 were collected and divided into 5 groups by periods of 5 years. The causes of cough had been primarily evaluated according to a step-by-step protocol and confirmed by the specific therapy. The changes in spectrum and frequency of causes were retrospectively analyzed by chi2 test. RESULTS: The common causes of chronic cough were cough variant asthma (n = 437, 46%), upper airway cough syndrome/postnasal drip syndrome (n = 304, 32%), eosinophilic bronchitis (n = 87, 9%), gastroesophageal reflux-related chronic cough (n = 83, 9%), postinfectious cough (n = 60, 6%) and angiotensin-converting enzyme inhibitors-induced cough (n = 46, 5%) in descending order. There were significant differences in the distribution and frequency of etiologies when analyzed by year (chi2 = 60.6, P = 0.0001). During a 5-year period, chronic cough due to cough variant asthma increased from 44% to 51% (chi2 = 12.8, P = 0.010), upper airway cough syndrome/postnasal drip syndrome decreased from 49% to 29% (chi2 = 20.1, P = 0.001), and gastroesophageal reflux increased from 2% to 10% (chi2 = 17.6, P = 0.002). However, chronic cough associated with eosinophilic bronchitis, postinfection and angiotensin-converting enzyme inhibitors remained stable. CONCLUSION: The common causes of chronic cough vary with time, which may have an impact on the strategy for the management of chronic cough.

Effects of phosphodiesterase 4 inhibitor on cough response in guinea pigs sensitized and challenged with ovalbumin.

BACKGROUND: There is currently considerable interest in the potential value of selective inhibitors of cyclic nucleotide phosphodiesterase 4 in the treatment of asthma. However, whether they influence eosinophilic airway inflammation-associated cough remains unclear. The objective of this study was to investigate the effects of selective phosphodiesterase 4 inhibitor SB207499 on cough response and airway inflammation in guinea pigs sensitized and challenged with ovalbumin. METHODS: Forty sensitized guinea pigs were randomly divided into four groups: control (n = 10), challenge (n = 10), SB207499 (n = 10) and aminophylline (n = 10), then challenged with aerosol of 1% ovalbumin or saline. Two hours later, animals were intraperitoneally injected with either saline, 25 mg/kg of SB207499 or aminophylline. At the 24th hour, the injection was repeated with 2.5 mg/kg and 25 mg/kg SB207499 or aminophylline, then cough response to inhaled capsaicin and airway responsiveness to methacholine inducing a 150% of the peak airway pressure to the baseline (PC150) was measured. Finally, total cell number and differentials in bronchoalveolar lavage fluid were analysed. RESULTS: The cough frequency per 3 minutes and PC150 in the challenge group were (22 +/- 4) times/3 minutes and (198 +/- 54) microg/ml, which were significantly different from (6 +/- 2) times/3 minutes and (691 +/- 81) microg/ml in the control group (P < 0.05, respectively). The injection of 25 mg/kg SB207499 significantly inhibited the increased cough response and airway hyperresponsiveness, the cough frequency and PC150 in guinea pigs were (13 +/- 2) times/3 minutes and (680 +/- 81) microg/ml (P < 0.05), which differed significantly from (18 +/- 2) times/3 minutes and (400 +/- 86) microg/ml after the administration of the same dose of aminophylline (P < 0.05). The inhibition of SB207499 on cough response was dose-dependent. Similarly, SB207499 decreased the total cell number and percentage of eosinophils in bronchoalveolar lavage fluid to (2.1 +/- 0.5) x 10(6)/ml and (20 +/- 5)% respectively, which were significantly different from (3.2 +/- 0.5) x 10(6)/ml and (29 +/- 5)% in the aminophylline group (P < 0.05, respectively) or (4.2 +/- 0.7) x 10(6)/ml and (35 +/- 4)% in the challenge group (P < 0.05, respectively). CONCLUSION: Phosphodiesterase 4 inhibitor may be more useful than aminophylline for cough associated with eosinophilic airway inflammation via inhibiting airway inflammation and airway hyperresponsiveness.

[The experimental study on the inhibitory effect of tripterine on airway inflammation in asthmatic mice].

OBJECTIVE: To investigate the effect and mechanism of tripterine on airway inflammation in asthmatic mice. METHODS: 30 BALB/c mice were randomly divided into a control group, an asthmatic group (ovalbumin-sensitized) and a tripterine (1 mg/kg, introperitoneally)-treated group. Pathologic changes in lung tissues, number of eosinophils in bronchoalveolar lavage fluid (BALF) and expression of stem cell factor (SCF) protein in lung were observed. In vitro, we established coculture system of bone marrow derived-mast cells from C57B6 mice and fibroblast NIH3T3, which were then treated by 2 micro mol/L of tripterine, and compared with NIH3T3 and mast cells. Concentration of histamine and eotaxin in supernants of coculture was measured by fluorometry and ELISA respectively, and expression of SCF protein in fibroblasts from cocultures was analyzed with immunohistochemistry. RESULTS: Compared with the asthmatic group, less inflammatory cell infiltration in lung tissues was observed in the tripterine-treated group. There was significant difference in the number of eosinophils in BALF between the tripterine-treated group [(0.56 +/- 0.03) x 10(6)/L] and the asthmatic group [(1.25 +/- 0.40) x 10(6)/L, P < 0.05]. So was the expression of SCF protein in lung tissue [0.74 +/- 0.20, 2.50 +/- 0.19, P < 0.01]. In vitro, the concentration of histamine and eotaxin in coculture supernants and the expression of SCF protein in fibroblasts from coculture were (3.83 +/- 0.41) ng/ml, (5.79 +/- 0.40) ng/ml and (95 +/- 3)%, respectively; after tripterine intervention, the data changed to (2.88 +/- 0.35) ng/ml, (4.24 +/- 0.29) ng/ml, (17 +/- 5)% (all P < 0.01). CONCLUSIONS: Tripterine might suppress airway inflammation in asthmatic mice, probably by downregulating the expression of SCF in fibroblasts, then inhibiting the production of histamine and eotaxin in mast cells.