[Investigation of diagnostic methods and criteria for gastroesophageal reflux-related cough].

Gastroesophageal reflux-related cough is a multidisciplinary disease that cannot be diagnosed solely based on typical reflux-related symptoms. Its current diagnostic methods and criteria are largely derived from those used for gastroesophageal reflux disease, with slight differences. Esophageal reflux monitoring can provide objective evidence for the diagnosis of gastroesophageal reflux-related cough and is therefore the first-choice of laboratory tests recommended by the guidelines for cough management. Acid exposure time and syndrome association probability have been accepted as the diagnostic criteria, while esophageal motility assessment also has some certain auxiliary diagnostic value. Based on the existing evidence, we have reviewed how to improve the diagnostic methods and criteria for gastroesophageal reflux-related cough, as well as the issues that need to be addressed in the future.

[Esophageal dysmotility detection in patients with gastroesophageal reflux-related cough and its influence on the efficacy of anti-reflux therapy].

Objective: To investigate the characteristics of esophageal dysmotility in patients with an initial diagnosis of acid/non-acid gastroesophageal reflux-related cough (GERC), and its correlation with the therapeutic response to anti-reflux treatments to search for the useful indicators to screen patients with chronic cough suitable for anti-reflux therapy. Methods: A total of 173 patients with suspicious GERC who attended the Chronic Cough Specialist Clinic of Department of Pulmonary and Critical Care Medicine, Tongji Hospital, School of Medicine, Tongji University between June 2020 and December 2022 were retrospectively selected for the study. The age of the patients was (45.1±14.6) years old, including 87 males and 86 females. Their demographic characteristics, clinical manifestations, and the results of high-resolution manometry (HRM) and multichannel intraluminal impedance-pH monitoring (MII-pH) were collected. Information on the etiological identification process and final diagnosis was also recorded. The recruited cases were grouped according to therapeutic outcomes and divided into cases with a favourable response to conventional anti-reflux treatment, cases with a favourable response to intensified anti-reflux treatment, and cases with no response to anti-reflux treatment. Factors influencing the efficacy of anti-reflux treatment were investigated. Differences between groups were compared using the χ2 test, Student-Newman-Keuls test, and Kruskal-Wallis H(K) test, where applicable. Logistic regression analysis using forward stepwise regression based on maximum likelihood estimation was used to screen for influence factors. Results: The 175 patients with suspicious GERC included 45 (26.0%) patients who responded to conventional anti-reflux treatment, 54 (31.2%) who responded to intensified anti-reflux treatment and 74 (42.8%) who did not respond to anti-reflux therapies. Esophageal dysmotility was present in 52.0% of patients (90/173), but was less common in patients who responded to conventional anti-reflux treatment (χ2=8.09, P=0.018). Although the majority of reflux episodes were non-acid (136/173, 78.6%), the proportion of acid reflux (χ2=19.49, P<0.001) and acid exposure time (H=11.04, P=0.004) were significantly higher in patients who responded to conventional anti-reflux treatment. The patients with acid and non-acid GERC had comparable proportion of esophageal dysmotility (64.9% vs. 48.5%, χ2=3.11, P=0.078), with a shorter break [2.4 (0.7, 5.6) cm vs. 6.1 (1.4, 10.0) cm, Z=-2.39, P=0.017], longer upper esophageal sphincter [(4.1±0.9) cm vs. (3.7±1.3) cm, t=-2.09, P=0.038], higher percentage of normal esophageal contractions [60.0% (17.8%, 90.0%) vs. 30.0% (0, 80.0%), Z=-2.14, P=0.032], and lower percentage of large break [10.0% (0, 40.0%) vs. 50.0% (0, 100.0%), Z=-2.92, P=0.004] in the patients with non-acid GERC. The mean resting pressure of the lower esophageal sphincter was significantly lower (H=7.49, P=0.024), while the percentage of ineffective esophageal contractions was markedly higher (H=8.60, P=0.014) in the patients who responded to intensified anti-reflux treatment and in the patients who did not respond to the anti-reflux therapies. Multifactorial logistic regression analysis identified the percentage of ineffective contraction as an independent factor predicting the efficacy of conventional anti-reflux treatment, with a cut-off value of≤45% and a moderate predictive value (AUC=0.67, P=0.004). Conclusions: Esophageal dysmotility is common in GERC patients with different characteristics in acid and non-acid GERC. The percentage of ineffective esophageal contraction may be a useful indicator for selecting an anti-reflux strategy and predicting treatment outcomes.

[Chronic cough in China: progress and perspectives].

Great progress has been made in regard of mechanism, etiological diagnosis and treatment of chronic cough since the beginning of the 20th century, and Chinese guidelines of diagnosis, management of cough was developed. Moreover, Chinese experts also participated in the formulation of international cough guidelines. Through the promotion of the guidelines, the level of diagnosis and treatment of chronic cough has been significantly improved in China. In the future, we should strengthen the studies on the epidemiology of chronic cough, the central regulation of chronic cough, and the treatment of refractory chronic cough, and promote the cough guidelines in primary community hospitals.

[Diagnosis and treatment of gastroesophageal reflux-related cough: consensus and controversies].

Typical reflux symptoms associated with gastroesophageal reflux-related cough have diagnostic implications. Questionnaires including these symptoms can be used as valuable screening tools for potential candidates. When reflux monitoring provides the objective evidence of abnormal reflux, the favorable response to anti-reflux therapy is essential for the diagnostic confirmation, in which anti-acid drugs are still the first-line choice. The benefits of neuromodulators in the treatment of refractory gastroesophageal reflux-related cough need to be further clarified. However, anti-reflux surgery may be a promising curative therapy for some patients with definitive surgical indications.

[Role and related mechanism of Mst-1 on regulating hypoxic reoxygenation induced autophagy and apoptosis in cardiomyocytes of mouse].

Objective: To explore the role and related mechanism of mammalian sterile 20-like kinase 1(Mst-1)in regulating hypoxia reoxygenation (HR) induced myocardial cell autophagy and apoptosis. Methods: Enzyme digestion method combined with differential adherent method was used to culture neonatal mouse myocardial cells. HR model was established by hypoxia for 24 hours and reoxygenation for 6 hours. The experimental groups including control group (normal cultured cardiomyocytes), Mst-1 empty virus group (cardiomyocytes transfected with recombinant lentiviral empty vector for 48 hours), Mst-1 knockdown group (recombinant lentivirus carrying Mst-1small interfering RNA (siRNA) was transfected into cardiomyocytes for 48 hours), Mst-1 overexpression group (cardiomyocytes were transfected with recombinant lentivirus carrying Mst-1 gene for 48 hours), HR group (cardiomyocytes exposed to HR), Mst-1 knockdown+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1siRNA) and Mst-1 overexpression+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1 gene). Real-time fluorescence quantitative RCR (qPCR) and Western blot were used to detect the relative expression of Mst-1 mRNA and protein in the cells, immunofluorescence staining was used to detect cardiomyocyte troponin T (cTnT), and autophagosomes and autophagy enzyme changes. TUNEL method was used to detect myocardial cell apoptosis, Western blot was adopted to detect autophagy-related protein microtubule-related protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, P62 and apoptosis-related protein cleaved-caspase 9, pro-caspase 9, cleaved-caspase-3, pro-caspase-3, and myeloid leukemia 1 (MCL-1) expression. MCL-1 inhibitor A1210477 was used to validate the signaling pathway of Mst-1 on regulating cardiomyocyte apoptosis and autophagy. Results: Immunofluorescence detection revealed that the cultured cells expressed cardiomyocyte-specific marker cTnT. The expression of Mst-1 in cardiomyocytes increased in HR model. Lentiviral transfection could effectively inhibit or overexpress Mst-1 in treated cells. The levels of autophagosomes and autophagolysosomes in cardiomyocytes undergoing HR and in Mst-1 overexpression+HR group were lower than those of control group, while autophagosomes and autophagolysosomes in cardiomyocytes of Mst-1 knockdown+HR group was significantly higher than in the HR group (all P<0.05). The TUNEL results showed that the proportion of TUNEL positive cells was significantly increased in the HR group and Mst-1 overexpression+HR group than in the control group, while the proportion of TUNEL positive cells was significantly decreased in the Mst-1 knockdown group+HR group as compared to the HR group (all P<0.05). Western blot results showed that the LC3 Ⅱ/LC3 Ⅰ levels were significantly lower, while the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly higher in the HR group and Mst-1 overexpression+HR group than in control group (all P<0.05). The LC3 Ⅱ/LC3 Ⅰ value was significantly higher, and the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly lower in the Mst-1 knockdown+HR group than in the HR group (P both<0.05). The expression level of P-MCL-1 protein was significantly lower in cardiomyocytes of HR and Mst-1 overexpression+HR group than in control group, and the expression level of P-MCL-1 protein was higher in Mst-1 knockdown+HR group than in HR group (P both<0.05). The recovery experiment showed that inhibiting MCL-1 in cells can block the regulatory effect of Mst-1 siRNA on cell autophagy and apoptosis. Conclusion: Inhibiting Mst-1 expression in cardiomyocytes can promote the autophagy of cardiomyocytes induced by hypoxic reoxygenation and reduce the apoptosis of cardiomyocytes via activating McL-1.

[Impact and related mechanism on the improvement of hyperglycemia-induced pyroptosis in H9c2 cells by mircoRNA-214].

Objective: To investigate whether microRNA(miR)-214 can improve hyperglycemia induced pyroptosis in H9c2 cells through targeting caspase-1. Methods: H9c2 cells of rats those in good growth condition were selected and incubated into the T25 culture bottle after digestion and passage. Cells were cultured in an incubator at 37 ℃ with 5%CO(2), repeat passage was made after cell density reached about 80%, The 5(th) to 8(th) generations of cells were selected for the subsequent experiments. To observe the effect of overexpression of miR-214 on pyroptosis and caspase-1 expression in H9c2 cells induced by hyperglycemia, the cells were divided into 4 groups: Control group(H9c2 cells cultured normally), Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimic-negative control+hyperglycaemic group(MNC+HG group, H9c2 cells were transfected with miR-214 mimic-negative control for 24 hours and then treated with 50 mmol/L hyperglycaemic for 24 hours). In order to further verify the anti-pyroptosis effect of miR-214 was mediated by targeted inhibition on caspase-1, cells overexpressing caspase-1 were used in the rescue experiment. The cells overexpressing caspase-1 were divided into 4 groups: Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimics+hyperglycosis+recombinant adenovirus (Ad-caspase-1-EGFP) group with caspase-1 gene and EGFP green fluorescent protein expression (mimics+HG+Ad-caspase-1-EGFP group, H9c2 cells were transfected with caspase-1-green fluorescent protein-carrying adenovirus for 48 hours, followed by transfection of miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycaemia for 24 hours), miR-214 mimics+HG+Ad-EGFP empty virus group (mimics+HG+Ad-EGFP group, H9c2 cells were transfected with empty adenovirus containing green fluorescent protein for 48 hours, followed by transfection with miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycosis for 24 hours). The mRNA expression levels of miRNA-214 and caspase-1 in cells were detected by real-time quantitative PCR. The expression and localization of caspase-1 protein were detected by immunofluorescence assay. Western blot was used to detect protein expression levels of procaspase-1, cleaved caspase-1, NLRP3 and ACS with ß-actin as internal reference. The secretion of IL-1ß and IL-18 in cell culture medium was detected by ELISA. The correlation between miR-214 and caspase-1 was detected by double luciferase reporter gene. Results: (1) The mRNA expression levels of miR-214 and caspase-1 in each group: the mRNA expressions of miR-214 in HG group and MNC+HG group were significantly lower than that in control group(P<0.05). The mRNA expression of miR-214 in mimics+HG group was significantly higher than that in control group (P<0.05). The mRNA expression levels of caspase-1 in HG group and MNC+HG group were significantly higher than that in control group(P<0.05). The mRNA expression level of caspase-1 in mimics+HG group was lower than that in control group(P<0.05). (2) The expression of caspase-1 in each group: the green fluorescence intensity in the control group was weak, which was strong in the HG group and MNC+HG group. The green fluorescence expression was weaker in mimics+HG group than in HG group. (3) ASC and NLRP3 protein expression levels in each group: ASC and NLRP3 protein expression levels in HG group and MNC+HG group were higher than those in control group(P<0.05). ASC and NLRP3 protein expression levels were significantly lower in mimics+HG group than in mimics+HG group (P<0.05). (4) The secretion of IL-1ß and IL-18 in the cell culture medium of each group: the content of IL-1ß and IL-18 in the cell culture medium of HG group and MNC+HG group was significantly higher than that of control group (P<0.05). The content of IL-1ß and IL-18 in the cell culture medium of mimics+HG group was significantly lower than that of the HG group (P<0.05). (5) Correlation between miR-214 and caspase-1: miR-214 specifically binds to caspase-1 3 'UTR. Meanwhile, Western blot results showed that cleaved caspase-1 protein expression levels were significantly higher in both HG group and MNC+HG group than in control group (P<0.05). The levels of cleaved caspase-1 were significantly lower in mimics+HG group than in HG group (P<0.05). There was no significant difference in procaspase-1 expression among groups (P>0.05). (6) The expression levels of procaspase-1, cleaved caspase-1, ASC and NLRP3 in each group in rescue experiment: there was no significant difference in the expression of procaspase-1 in each group (P>0.05). Cleaved caspase-1, ASC and NLRP3 protein expressions were significantly lower in mimics+HG group than in HG group (P<0.05). However, cleaved caspase-1, ASC and NLRP3 protein expressions were significantly higher in mimics+HG+ Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). (7) The expression of IL-1ß and IL-18 in rescue experiment: the secretions of IL-1ß and IL-18 in the cell culture medium of the mimics+HG group were significantly lower than that of HG group (P<0.05), which were significantly higher in mimics+HG+Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). Conclusion: miR-214 can improve the hyperglycemia induced pyroptosis in H9c2 cells by targeted inhibition of the caspase-1.

[Changes and implications of esophageal function in patients with chronic cough induced by gastroesophageal reflux].

Objective: To explore the changes in the esophageal function and their association with the therapeutic outcome in patients with gastroesophageal reflux-induced chronic cough (GERC). Methods: One hundred thirty-five patients with definite GERC consecutively referred to our respiratory clinic were recruited into the study between January 2012 and August 2015.Cough was due to acid reflux in 81 patients and non-acid reflux in 54 patients, with the favorable response to the standard antireflux therapy in 88 patients and to the intensified antireflux treatment in 47 patients. The control groups included 26 patients with gastroesphageal reflux disease without cough and 22 healthy volunteers. All the subjects underwent an esophageal manometry from which the parameters were recorded, including the pressure, length and relaxation rate of lower esophageal sphincter, and the peristaltic contractive amplitude, wave velocity and contractive time of esophagus. The data were combined with the results of multi-channel intraluminal impedance combined with pH monitoring to analyze the changes of esophageal function in the patients with acid or non-acid GERC and their relation to the outcomes of antireflux therapy. Results: Compared with healthy volunteers, patients with GERC presented with a lower pressure [(11±5) mmHg vs (15±5) mmHg (1 mmHg=0.133 kPa), q=3.70, P=0.000], shorter overall length [(2.2±0.5) cm vs (3.0±1.0) cm, q=2.78, P=0.017] and similar relaxation rate of lower esophageal sphincter(q=1.14, P=0.258). Furthermore, they also showed a decrease in esophageal peristaltic contractive amplitude [(33±13) mmHg vs (45±11) mmHg, q=2.19, P=0.030] and wave velocity [(2.6±0.8) cm/s vs (3.4±0.6) cm/s, q=2.91, P=0.010] but an increase in esophageal contractive time of esophagus [(4.9±2.2) s vs (3.1±0.8) s, q=3.25, P=0.001] in addition to a linear negative correlation between esophageal peristaltic wave velocity and bolus clearance (r=-0.603, P=0.000). However, these parameters were not different between patients with GERC and gastroesophageal reflux disease without cough. The patients with GERC due to acid and non-acid reflux presented with a similar esophageal dysmotility but different variables reflecting the acidity of refluxates as indicated by multi-channel intraluminal impedance combined with pH monitoring. The esophageal peristaltic wave velocity was significantly lower in the patients with GERC responsive to the standard antireflux therapy than in those responsive to the intensified antireflux therapy [(2.2±0.6) cm/s vs (3.0±1.0) cm/s, t= 2.066, P= 0.041]. Conclusions: Esophageal dysfunction is present in patients with GERC. Its characteristics and severity are not associated with the types of gastroesophageal reflux inducing cough, but may predict the efficacy of medical antireflux therapy.

[Validation of the Chinese version of Hull airway reflux questionnaire and its application in the evaluation of chronic cough].

OBJECTIVE: To validate the effectiveness, repeatability and treatment responsiveness of the Chinese version of Hull airway reflux questionnaire (HARQ), and to determine its clinical value. METHODS: A standard Chinese version of HARQ was developed by an established translation procedure and its repeatability was assessed in a preliminary study involving 55 untreated patients with stable chronic cough. Thereafter, a total of 132 patients with chronic cough referred to our respiratory clinic were recruited into the study between May 2014 and April 2015. After their cough was evaluated with the HARQ, cough symptom score or cough reflex sensitivity to inhaled capsaicin and the correlations among them were analyzed, and the causes of chronic cough in all the patients were presumptively determined according to an established diagnostic protocol and finally confirmed with the subsequent therapy specific to the etiologies. After two weeks of effective treatment, HARQ, cough symptom score and cough reflex sensitivity to inhaled capsaicin were measured once more, and treatment responsiveness was calculated. The score of the HARQ in 132 patients with chronic cough were compared with that in 104 healthy volunteers. RESULTS: The repeatability of the Chinese version of the HARQ was validated at a week interval with the intraclass correlation coefficients of 0.96 (95%CI: 0.93-0.98, P=0.00) for total score (n=55). No correlation was found between the HARQ and cough symptom score, and the HARQ showed a weak negative correlation with cough threshold C2 (rIgC2=-0.23, P=0.01) but not with C5. The value of HARQ was significantly higher in patients with chronic cough than in healthy volunteers[20.00(14.00, 28.00) vs 4.00 (2.00, 6.00), Z=-12.89, P=0.00], but no difference of HARQ in gender or age existed in chronic cough. Although all the patients with various etiologies of chronic cough had an increased value of HARQ, cough due to gastroesophageal reflux showed the highest HARQ score among them[28.50 (25.00, 32.25) vs 18.50 (14.00, 26.25), Z=4.43, P=0.00]. After two weeks of effective treatment, the HARQ score decreased from 20.00 (14.00, 28.00) pre-treatment to 10.00 (4.25, 17.75) post-treatment (Z=-6.06, P=0.00), with 52.04% of score change ratio, 1.38 of effect side and 1.97 of standard response mean respectively. CONCLUSION: HARQ is a reliable and valid tool for the management of chronic cough with good treatment responsiveness, and may be used as an easy way to predict cough due to gastroesophageal reflux.

Molecular characterization of a novel form of (A gamma delta beta)zero thalassemia deletion in a Chinese family.

We have identified and molecularly characterized a novel deletion in the beta-globin gene cluster that is associated with elevated fetal hemoglobin in the adult. The propositus is a homozygote from the Yunnan province of China. The deletion spans about 90 kb of DNA and removes the A gamma, delta, and beta-globin genes. The 5' breakpoint of the deletion is located about 0.13 kb upstream from the A gamma-globin gene, whereas the 3' breakpoint is located about 66 kb downstream from the beta-globin gene, about 13 kb upstream from the breakpoint of the Chinese (A gamma delta beta)zero-thalassemia. Heterozygotes for this Yunnanese form of (A gamma delta beta)zero-thalassemia express between 9% and 17% of fetal hemoglobin, whereas the homozygote present with a mild anemia (Hb = 10.7 g/dl). Comparison of the sites of 3' breakpoints of the Yunnanese and the Chinese (A gamma delta beta)zero-thalassemia mutants is compatible with the hypothesis that an enhancer element is located between the 3' breakpoints of these two mutants. Juxta-position to the G gamma gene of this element may be responsible for the efficient gamma-gene expression in the Yunnanese mutant.

Ultrastructure of the secondary tympanic membrane in the human fetus.

The normal secondary tympanic membrane in human fetuses was examined by transmission electron microscopy. The membranes in 5- to 9-month-old fetuses consist of the following three layers: (1) an outer squamous epithelial layer facing the middle ear, which is not formed until 4 months old; (2) a middle fibrous layer containing collagen, elastin, fibroblasts and fibrocytes, and (3) an inner layer of flat cells facing the scala tympani. Following the maturation of the fetus the epithelium is getting thinner and fibroblasts are reduced in number, but fibrocytes are increased and collagen and elastin grow gradually in density. The ultrastructure of the secondary tympanic membrane at 8 month is mature in type and shows the same characteristics as in the adult. This membrane has an important and complicated physiological function. The epithelium of the outer layer, with tight junctions and multiple desmosomes, provides a barrier to keep harmful substances out. The stability of the membrane provides protection against rupture, while the elasticity plays a role in the physiology of hearing as well.