Cellular senescence-Related genes define the immune microenvironment and molecular characteristics in severe asthma patients.

Recent studies have shown that cellular senescence is involved in the pathogenesis of severe asthma (SA). The objective of this study was to investigate the role of cellular senescence-related genes (CSGs) in the pathogenesis of SA. Here, 54 differentially expressed CSGs were identified in SA patients compared to healthy control individuals. Among the 54 differentially expressed CSGs, 3 CSGs (ETS2, ETS1 and AURKA) were screened using the LASSO regression analysis and logistic regression analysis to establish the CSG-based prediction model to predict severe asthma. Moreover, we found that the protein expression levels of ETS2, ETS1 and AURKA were increased in the severe asthma mouse model. Then, two distinct senescence subtypes of SA with distinct immune microenvironments and molecular biological characteristics were identified. Cluster 1 was characterized by increased infiltration of immature dendritic cells, regulatory T cells, and other cells. Cluster 2 was characterized by increased infiltration levels of eosinophils, neutrophils, and other cells. The molecular biological characteristics of Cluster 1 included aerobic respiration and oxidative phosphorylation, whereas the molecular biological characteristics of Cluster 2 included activation of the immune response and immune receptor activity. Then, we established an Random Forest model to predict the senescence subtypes of SA to guide treatment. Finally, potential drugs were searched for each senescence subgroup of SA patients via the Connectivity Map database. A peroxisome proliferator-activated receptor agonist may be a potential therapeutic drug for patients in Cluster 1, whereas a tachykinin antagonist may be a potential therapeutic drug for patients in Cluster 2. In summary, CSGs are likely involved in the pathogenesis of SA, which may lead to new therapeutic options for SA patients.

Randomised, controlled crossover trial of intermittent and continuous transcutaneous electrical stimulation of the genioglossus muscle for obstructive sleep apnoea.

PURPOSE: Continuous transcutaneous electrical stimulation (CTES) of the genioglossus muscle may benefit patients with obstructive sleep apnoea (OSA). However, the therapeutic value of intermittent transcutaneous electrical stimulation (ITES) for OSA is unclear. METHODS: This was a randomised, controlled, crossover study to compare the effects of ITES and CTES of the genioglossus muscle. Over three single-night sessions, participants were alternately subjected to three genioglossus stimulation modalities during sleep (sham, CTES and ITES). The apnoea-hypopnoea index (AHI) and oxygen desaturation index (ODI) were used for OSA diagnosis and to evaluate efficacy. A responder was defined as an individual with a ≥50% reduction in AHI together with <10 AHI events per hour and/or an ODI reduction of ≥25% between sham stimulation and electrical stimulation nights. RESULTS: Fifteen men with OSA completed the study. Compared with sham, the median AHI with ITES decreased by 13.3 events/hour (95% CI 3.1 to 23.5, p=0.030) and by 7.3 events/hour (95% CI -3.9 to 18.5, p=0.825) with CTES. The median ODI was reduced by 9.25 events/hour (95% CI 0.5 to 18.0) with ITES and 3.3 events/hour (95% CI -5.6 to 12.2) with CTES; however, there was no significant difference between groups. Furthermore, ITES outperformed CTES with respect to longest apnoea duration (median (95% CI), 9.5 (0.0 to 19.0), p=0.011)) and the highest sleep efficiency (12.2 (2.7 to 21.7), p=0.009). Of the 15 participants, 8 responded to ITES and 3 responded to CTES (p=0.058), of whom all eight cases and two out of three cases had ODIs <5 events/hour, respectively. All participants tolerated ITES well. CONCLUSIONS: ITES improved upper airway obstruction in patients with OSA, suggesting that further prospective validation of the intermittent approach is warranted. TRIAL REGISTRATION NUMBER: ChiCTR2100050138.

Identification of hub genes and potential biomarkers of neutrophilic asthma: evidence from a bioinformatics analysis.

OBJECTIVE: Asthma is a chronic airway inflammatory disease caused by multiple genetic and environmental factors. This study mainly sought to provide potential therapeutic targets and biomarkers for neutrophilic asthma (NA). METHODS: Three gene expression profiling datasets were obtained from the Genome Expression Omnibus (GEO) database. GSE45111 and GSE41863 were used to identify hub genes and potential biomarkers, and GSE137268 was used for data verification. We verified the repeatability of intragroup data and identified differentially expressed genes (DEGs). Then, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs, and a protein-protein interaction (PPI) network was constructed to identify the hub genes. Finally, receiver operating characteristic (ROC) analysis was used to verify the ability of the hub genes to differentiate between NA and eosinophilic asthma (EA). RESULTS: In this study, we identified 411 DEGs by comprehensive analysis of NA/EA patients and NA/healthy controls (HCs). Ten hub genes (CXCR1, FCGR3B, CXCR2, SELL, S100A12, CSF3R, IL6R, JAK3, CD48, and GNG2) were identified from the PPI network. Finally, based on the ROC analysis, 7 genes showed good diagnostic value for discriminating NA from EA-CXCR1, FCGR3B, CXCR2, SELL, S100A12, CSF3R, and IL6R (AUC > 0.7). CONCLUSION: We identified 7 hub genes that can distinguish NA from EA. The IL-8-mediated signaling may be the primary pathway to determine the NA phenotype in asthma. CXCR1/2 and S100A12 may be the primary genes determining the NA phenotype. CXCR1/2 and S100A12 might be biomarkers and new therapeutic targets for NA.Supplemental data for this article is available online at at.

Cross-talk of four types of RNA modification writers defines the immune microenvironment in severe asthma.

Adenine modifications, including m6 A, m1 A, APA, and A-to-I modifications, are the most impactful RNA modifications. These modifications are primarily produced by enzymes called writers. The main purpose of this study was to explore the cross-talk and potential roles of these writers in severe asthma. We found 13 RNA writers potentially related to severe asthma and three RNA modification patterns. Cluster 3 showed predominant neutrophil infiltration and C-type lectin receptor signaling; cluster 1 showed predominant innate immune cell infiltration and ubiquitin-proteasome system activation; and cluster 2 did not show obvious immune infiltration characteristics. We found that RNA modification writers modified immune cell-related genes and led to both accumulation of different immune cells in the airways and activation of a series of biological processes, which ultimately leads to severe asthma. TRMT6, WTAP, and TRMT6A were included in a random forest model as predictors. Cromoglicic acid, thioperamide, and fluvastatin were potential drugs for clusters 1, 2, and 3, respectively. We found that cross-talk of RNA modifications is significant in severe asthma, which provides insight into severe asthma pathogenesis and possible treatment avenues.

MicroRNA-98-5p Inhibits IL-13-Induced Proliferation and Migration of Human Airway Smooth Muscle Cells by Targeting RAC1.

The dysfunction of airway smooth muscle cells (ASMCs) is one of the key factors in the pathogenesis of asthma. How miR-98-5p works in asthma has not been completely elucidated. This work focused on how miR-98-5p functions in the proliferation and migration of human ASMCs treated with interleukin-13 (IL-13). MiR-98-5p expression in plasma of asthmatic patients and IL-13-stimulated ASMCs was probed by quantitative real-time polymerase chain reaction (qRT-PCR). RAS-relevant C3 botulinum toxin substrate 1 (RAC1) protein expression in ASMCs was assessed by Western blot. The growth of ASMCs was measured by cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. The migration of ASMCs was examined by Transwell assay. Besides, the apoptosis of ASMCs was analyzed by flow cytometry. The targeting relationship between miR-98-5p and RAC1 3'-UTR was verified by a dual-luciferase reporter gene assay. MiR-98-5p expression was reduced in patients' plasma and IL-13-stimulated ASMCs, and RAC1 expression was upregulated in ASMCs treated with IL-13. MiR-98-5p overexpression inhibited IL-13-induced proliferation and migration of ASMCs while promoting the apoptosis. The opposite result was observed after inhibiting miR-98-5p expression. Besides, RAC1 was identified as a direct downstream target of miR-98-5p in ASMCs. The restoration of RAC1 expression counteracted the impacts of miR-98-5p overexpression on IL-13-stimulated proliferation, migration, and apoptosis of ASMCs. MiR-98-5p inhibits IL-13-induced proliferation and migration and accelerates the apoptosis of ASMCs by downregulating RAC1 expression.

MicroRNA-15b-5p inhibits tumor necrosis factor alpha-induced proliferation, migration, and extracellular matrix production of airway smooth muscle cells via targeting yes-associated protein 1.

The excessive proliferation and the deposition of extracellular matrix (ECM) of airway smooth muscle (ASM) cells facilitates airway remodeling in asthma. This study explores how microRNA-15b-5p (miR-15b-5p) functions in modulating the proliferation, migration, inflammatory response, and ECM deposition of ASM cells. MiR-15b-5p and yes-associated protein 1 (YAP1) mRNA expression levels in tumor necrosis factor alpha (TNF-α)-induced ASM cells were, respectively, examined by real-time quantitative polymerase-chain reaction. Besides, the proliferative ability and migrative potential of ASM cells were examined by cell counting kit-8 assay, 5-bromo-2 '-deoxyuridine assay, and transwell assays, respectively. Interleukin-6 and interleukin-8 levels in ASM cells were detected by enzyme-linked immunosorbent assay. YAP1, collagen I, and collagen III expressions in ASM cells were detected by Western blot. With dual-luciferase reporter gene assay, the relations between miR-15b-5p and YAP1 3'UTR in ASM cells was examined. MiR-15b-5p expression level was reduced in ASM cells treated with TNF-α. MiR-15b-5p repressed TNF-α-initiated growth and migration of ASM cells and also suppressed IL-6 and IL-8 secretion, and inhibited collagen I and collagen III expressions in ASM cells. Furthermore, it was validated that YAP1 was a downstream target of miR-15b-5p in ASM cells. Notably, YAP1 overexpression attenuated the inhibitory effects of miR-15b-5p up-regulation on the proliferation, migration, and inflammatory response, as well as ECM deposition of TNF-α-induced ASM cells. In conclusion, miR-15b-5p/YAP1 axis modulates the growth, migration, inflammatory response, and ECM deposition of ASM cells, thus participating in the pathogenesis of asthma.

Diagnostic value of using a combination of nucleic acid and specific antibody tests for SARS-CoV-2 in coronavirus disease 2019.

Coronavirus disease 2019 (COVID-19) is a newly emerged disease with various clinical manifestations and imaging features. The diagnosis of COVID-19 depends on a positive nucleic acid amplification test by real-time reverse transcription-polymerase chain reaction (RT-PCR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the clinical manifestations and imaging features of COVID-19 are non-specific, and nucleic acid test for SARS-CoV-2 can have false-negative results. It is presently believed that detection of specific antibodies to SARS-CoV-2 is an effective screening and diagnostic indicator for SARS-CoV-2 infection. Thus, a combination of nucleic acid and specific antibody tests for SARS-CoV-2 will be more effective to diagnose COVID-19, especially to exclude suspected cases.

Treatment of Coronavirus Disease 2019 Patients With Prolonged Postsymptomatic Viral Shedding With Leflunomide: A Single-center Randomized Controlled Clinical Trial.

BACKGROUND: We aimed to evaluate the efficacy and safety of leflunomide, an approved dihydroorotate dehydrogenase inhibitor, to treat coronavirus disease 2019 (COVID-19) patients with prolonged postsymptomatic viral shedding. METHODS: We conducted a prospective, randomized controlled, open-label trial involving hospitalized adult COVID-19 patients with prolonged polymerase chain reaction (PCR) positivity. Patients were randomly assigned to receive either leflunomide (50 mg every 12 hours, 3 consecutive times, orally; then 20 mg once daily for 8 days), in addition to nebulized interferon alpha 2a (IFN-α-2a, 3 million IU each time, twice daily for 10 days), or nebulized IFN-α-2a alone for 10 days. The primary endpoint was the duration of viral shedding. RESULTS: A total of 50 COVID-19 patients with prolonged PCR positivity were randomized into 2 groups: 26 were assigned to the leflunomide plus IFN-α-2a group, and 24 were assigned to the interferon-alone group. Treatment with leflunomide was not associated with a difference from the interferon-alone group in the duration of viral shedding (hazard ratio for negative reverse-transcription PCR, 0.70 [95% confidence interval, .391-1.256]; P = .186). In addition, the patients given leflunomide did not have a substantially shorter length of hospital stay than patients treated with interferon alone, with median durations of 29.0 (interquartile range [IQR], 19.3-47.3) days and 33.0 (IQR, 29.3-42.8) days, respectively (P = .170). Two leflunomide recipients were unable to complete the full 10-day course of administration due to adverse events. CONCLUSIONS: In COVID-19 patients with prolonged PCR positivity, no benefit in terms of the duration of viral shedding was observed with the combined treatment of leflunomide and IFN-α-2a beyond IFN-α-2a alone.

Asymptomatic infection by SARS-CoV-2 in healthcare workers: A study in a large teaching hospital in Wuhan, China.

OBJECTIVES: To investigate the proportion and characteristics of asymptomatic infection among healthcare workers (HCWs). METHODS: This study retrospectively investigated 1407 HCWs who were screened for COVID-19 by chest computed tomography (CT) scans and nasopharyngeal swabs for SARS-CoV-2 nucleic acid. Demographics, CT features, nasopharyngeal swabs, baseline symptoms, and laboratory data were collected. RESULTS: Of 1407 HCWs, 235 had symptoms and 1172 were asymptomatic close contacts, of which, 107 were symptomatic cases and 84 were close contacts who had abnormal CT findings. Of 152 symptomatic individuals and 908 close contacts tested for SARS-CoV-2 nucleic acid, 122 symptomatic cases and 38 close contacts had positive reverse-transcriptase real-time polymerase chain (RT-PCR) test results. The rate of confirmed asymptomatic infections was 4.2% (38/908). Both symptomatic and asymptomatic infected cases had high titrations of specific IgG or had ≥four-fold increase in IgG during convalescence compared with the acute phase. Combining the RT-PCR tests and serological findings, the rate of asymptomatic infections was 9.7% (88/908). In terms of the duration of viral shedding, there was no significant difference between symptomatic mild/moderate participants and asymptomatic infections. CONCLUSIONS: The findings demonstrated that a high rate of asymptomatic SARS-CoV-2 carriers existed among healthcare worker close contacts during the outbreak of COVID-19.

Abnormal immunity of non-survivors with COVID-19: predictors for mortality.

BACKGROUND: The number of coronavirus disease 2019 (COVID-19) cases has rapidly increased all over the world. Specific information about immunity in non-survivors with COVID-19 is scarce. This study aimed to analyse the clinical characteristics and abnormal immunity of the confirmed COVID-19 non-survivors. METHODS: In this single-centered, retrospective, observational study, we enrolled 125 patients with COVID-19 who were died between January 13 and March 4, 2020 in Renmin Hospital of Wuhan University. A total of 414 randomly recruited patients with confirmed COVID-19 who were discharged from the same hospital during the same period served as control. The demographic, clinical characteristics and laboratory findings at admission, and treatment used in these patients were collected. The immunity-related risk factors associated with in-hospital death were tested by logistic regression models and Receiver Operating Characteristic (ROC) curve. RESULTS: Non-survivors (70 years, IQR: 61.5-80) were significantly older than survivors (54 years, IQR: 37-65) (P <  0.001). 56.8% of non-survivors was male. Nearly half of the patients (44.9%) had chronic medical illness. In non-survivors, hypertension (49.6%) was the most common comorbidity, followed by diabetes (20.0%) and coronary heart disease (16.0%). The common signs and symptoms at admission of non-survivors were fever (88%), followed by cough (64.8%), dyspnea (62.4%), fatigue (62.4%) and chest tightness (58.4%). Compared with survivors, non-survivors had higher white blood cell (WBC) count (7.85 vs 5.07 × 109/L), more elevated neutrophil count (6.41 vs 3.08 × 109/L), smaller lymphocyte count (0.69 vs 1.20 × 109/L) and lower platelet count (172 vs 211 × 109/L), raised concentrations of procalcitonin (0.21 vs 0.06 ng/mL) and CRP (70.5 vs 7.2 mg/L) (P < 0.001). This was accompanied with significantly decreased levels of CD3+ T cells (277 vs 814 cells/µl), CD4+ T cells (172 vs 473 cells/µl), CD8+ T cells (84 vs 262.5 cells/µl, P < 0.001), CD19+ T cells (88 vs 141 cells/µl) and CD16+ 56+ T cells (79 vs 128.5 cells/µl) (P < 0.001). The concentrations of immunoglobulins (Ig) G (13.30 vs 11.95 g/L), IgA (2.54 vs 2.21 g/L), and IgE (71.30 vs 42.25 IU/ml) were increased, whereas the levels of complement proteins (C)3 (0.89 vs 0.99 g/L) and C4 (0.22 vs 0.24 g/L) were decreased in non-survivors when compared with survivors (all P < 0.05). The non-survivors presented lower levels of oximetry saturation (90 vs 97%) at rest and lactate (2.40 vs 1.90 mmol/L) (P < 0.001). Old age, comorbidity of malignant tumor, neutrophilia, lymphocytopenia, low CD4+ T cells, decreased C3, and low oximetry saturation were the risk factors of death in patients with confirmed COVID-19. The frequency of CD4+ T cells positively correlated with the numbers of lymphocytes (r = 0.787) and the level of oximetry saturation (r = 0.295), Whereas CD4+ T cells were negatively correlated with age (r =-0.323) and the numbers of neutrophils (r = - 0.244) (all P < 0.001). CONCLUSIONS: Abnormal cellular immunity and humoral immunity were key features of non-survivors with COVID-19. Neutrophilia, lymphocytopenia, low CD4+ T cells, and decreased C3 were immunity-related risk factors predicting mortality of patients with COVID-19.

A Small-Scale Medication of Leflunomide as a Treatment of COVID-19 in an Open-Label Blank-Controlled Clinical Trial.

We recently reported that inhibitors against human dihydroorotate dehydrogenase (DHODH) have broad-spectrum antiviral activities including their inhibitory efficacies on SARS-CoV-2 replication in infected cells. However, there are limited data from clinical studies to prove the application of DHODH inhibitors in Coronavirus disease 2019 (COVID-19) patients. In the present study, we evaluated Leflunomide, an approved DHODH inhibitor widely used as a modest immune regulator to treat autoimmune diseases, in treating COVID-19 disease with a small-scale of patients. Cases of 10 laboratory-confirmed COVID-19 patients of moderate type with obvious opacity in the lung were included. Five of the patients were treated with Leflunomide, and another five were treated as blank controls without a placebo. All the patients accepted standard supportive treatment for COVID-19. The patients given Leflunomide had a shorter viral shedding time (median of 5 days) than the controls (median of 11 days, P = 0.046). The patients given Leflunomide also showed a significant reduction in C-reactive protein levels, indicating that immunopathological inflammation was well controlled. No obvious adverse effects were observed in Leflunomide-treated patients, and they all discharged from the hospital faster than controls. This preliminary study on a small-scale compassionate use of Leflunomide provides clues for further understanding of Leflunomide as a potential antiviral drug against COVID-19.

Clinical features in 52 patients with COVID-19 who have increased leukocyte count: a retrospective analysis.

Recent reports have showed that a proportion of patients with Coronavirus Disease 2019 (COVID-19) presented elevated leukocyte count. Clinical data about these patients is scarce. We aimed to evaluate the clinical findings of patients with COVID-19 who have increased leukocyte at admission. We retrospectively collected the clinical data on the 52 patients who have increased leukocyte count at admission from the 619 patients with confirmed COVID-19 who had pneumonia with abnormal features on chest CT scan in Renmin Hospital of Wuhan University in Wuhan, China, from February 3 to March 3, 2020. The mean age of the 52 patients with increased leukocyte count was 64.7 (SD 11.4) years, 32 (61.5%) were men and 47 (90.4%) had fever. Compared with the patients with non-increased leukocyte count, the patients with increased leukocyte count were significantly older (P < 0.01), were more likely to have underlying chronic diseases (P < 0.01), more likely to develop critically illness (P < 0.01), more likely to admit to an ICU (P < 0.01), more likely to receive mechanical ventilation (P < 0.01), had higher rate of death (P < 0.01) and the blood levels of neutrophil count and the serum concentrations of CRP and IL-6 were significantly increased, (P < 0.01). The older patients with COVID-19 who had underlying chronic disorders are more likely to develop leukocytosis. These patients are more likely to develop critical illness, with a high admission to an ICU and a high mortality rate.