Single-cell analysis of diverse immune phenotypes in malignant pleural effusion.

The complex interactions among different immune cells have important functions in the development of malignant pleural effusion (MPE). Here we perform single-cell RNA sequencing on 62,382 cells from MPE patients induced by non-small cell lung cancer to describe the composition, lineage, and functional states of infiltrating immune cells in MPE. Immune cells in MPE display a number of transcriptional signatures enriched for regulatory T cells, B cells, macrophages, and dendritic cells compared to corresponding counterparts in blood. Helper T, cytotoxic T, regulatory T, and T follicular helper cells express multiple immune checkpoints or costimulatory molecules. Cell-cell interaction analysis identifies regulatory B cells with more interactions with CD4+ T cells compared to CD8+ T cells. Macrophages are transcriptionally heterogeneous and conform to M2 polarization characteristics. In addition, immune cells in MPE show the general up-regulation of glycolytic pathways associated with the hypoxic microenvironment. These findings show a detailed atlas of immune cells in human MPE and enhance the understanding of potential diagnostic and therapeutic targets in advanced non-small cell lung cancer.

Prolonged SARS-CoV-2 Viral Shedding in Patients with COVID-19 was Associated with Delayed Initiation of Arbidol Treatment and Consulting Doctor Later: A Retrospective Cohort Study.

OBJECTIVE: To study data about SARS-CoV-2 virus shedding and clarify the risk factors for prolonged virus shedding. METHODS: Data were retrospectively collected from adults hospitalized with laboratory-confirmed coronavirus disease-19 (COVID-19) in Wuhan Union Hospital. We compared clinical features among patients with prolonged (a positive SARS-CoV-2 RNA on day 23 after illness onset) and short virus shedding and evaluated risk factors associated with prolonged virus shedding by multivariate regression analysis. RESULTS: Among 238 patients, the median age was 55.5 years, 57.1% were female, 92.9% (221/238) were administered with arbidol, 58.4% (139/238) were given arbidol in combination with interferon. The median duration of SARS-CoV-2 virus shedding was 23 days (IQR, 17.8-30 days) with a longest one of 51 days. The patients with prolonged virus shedding had higher value of D-dimer (P=0.002), IL-6 (P<0.001), CRP (P=0.005) and more lobes lung lesion (P=0.014) on admission, as well as older age (P=0.017) and more patients with hypertension (P=0.044) than in those the virus shedding less than 23 days. Multivariate regression analysis revealed that prolonged viral shedding was significantly associated with initiation arbidol >8 days after symptom onset [OR: 2.447, 95% CI (1.351-4.431)], ≥3 days from onset of symptoms to first medical visitation [OR: 1.880, 95% CI (1.035-3.416)], illness onset before Jan. 31, 2020 [OR: 3.289, 95% CI (1.474-7.337)]. Arbidol in combination with interferon was also significantly associated with shorter virus shedding [OR: 0.363, 95% CI (0.191-0.690)]. CONCLUSION: Duration of SARS-CoV-2 virus shedding was long. Early initiation of arbidol and arbidol in combination with interferon as well as consulting doctor timely after illness onset were helpful for SARS-CoV-2 clearance.

Pleural effusion as an indicator for the poor prognosis of COVID-19 patients.

BACKGROUND: Coronavirus Disease 19 (COVID-19) is a global health concern that has become a pandemic over the past few months. This study aims at understanding the clinical manifestations of COVID-19 patients with pleural effusion. METHODS: COVID-19 patients were retrospectively enrolled from the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Pharyngeal swabs from patients were tested using real-time polymerase chain reaction. Patients with COVID-19 were divided into two groups based on their computed tomography (CT) scans for the presence of pleural effusion at admission. We compared the clinical features, laboratory findings, scans and clinical outcomes between the two groups. RESULTS: Pleural effusion was observed in 9.19% of the patients. Patients with pleural effusion were more likely to be severe or critical cases. Moreover, patients with pleural effusion were associated with increased mortality. Of the 799 discharged patients, patients with pleural effusion had longer hospital stays and duration of viral shedding since the onset of symptoms as compared with that for patients without pleural effusion. After discharge, 217 patients visited for a follow-up CT re-examination at the Union Hospital. The CT scans showed that patients with pleural effusion required a longer time to resolve the lung inflammation after the onset of COVID-19 as compared with the time required by patients without pleural effusion. CONCLUSION: This population of patients requires special attention and pleural effusion may be an indicator of poor prognosis in COVID-19 patients.

Corticosteroids Treatment of Patients with Coronavirus Disease 2019: A Propensity Score Matching Study.

The role of corticosteroids in the treatment of Coronavirus disease 2019 (COVID-19) is controversial. In the present study, we evaluated the effects of adjuvant corticosteroids treatment on the outcome of patients with COVID-19 (n=966), using Propensity Score Matching to adjust for potential differences between the corticosteroids group (n=289) and the non-corticosteroids group (n=677). Analysis of data without adjusting differences in baseline characteristics indicated that the proportion of mechanical ventilation and the mortality was higher in the corticosteroids treatment group in total or severe/critical patients. The duration of viral shedding was longer in the non-corticosteroids treatment group in total or general/mild patients. After adjusting the difference between the corticosteroids and non-corticosteroids treatment group, the analysis revealed that the use of corticosteroids had no effect on the duration of viral shedding, in-hospital mortality or 28-day mortality.

Early Use of Corticosteroid May Prolong SARS-CoV-2 Shedding in Non-Intensive Care Unit Patients with COVID-19 Pneumonia: A Multicenter, Single-Blind, Randomized Control Trial.

BACKGROUND: There is still no clinical evidence available to support or to oppose corticosteroid treatment for coronavirus disease 2019 (COVID-19) pneumonia. OBJECTIVE: To investigate the efficacy and safety of corticosteroid given to the hospitalized patients with COVID-19 pneumonia. METHODS: This was a prospective, multicenter, single-blind, randomized control trial. Adult patients with COVID-19 pneumonia who were admitted to the general ward were randomly assigned to either receive methylprednisolone or not for 7 days. The primary end point was the incidence of clinical deterioration 14 days after randomization. RESULTS: We terminated this trial early because the number of patients with COVID-19 pneumonia in all the centers decreased in late March. Finally, a total of 86 COVID-19 patients underwent randomization. There was no difference of the incidence of clinical deterioration between the methylprednisolone group and control group (4.8 vs. 4.8%, p = 1.000). The duration of throat viral RNA detectability in the methylprednisolone group was 11 days (interquartile range, 6-16 days), which was significantly longer than that in the control group (8 days [2-12 days], p = 0.030). There were no significant differences between the 2 groups in other secondary outcomes. Mass cytometry discovered CD3+ T cells, CD8+ T cells, and NK cells in the methylprednisolone group which were significantly lower than those in the control group after randomization (p < 0.05). CONCLUSIONS: From this prematurely closed trial, we found that the short-term early use of corticosteroid could suppress the immune cells, which may prolong severe acute respiratory syndrome coronavirus 2 shedding in patients with COVID-19 pneumonia. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04273321.

A cluster of health care workers with COVID-19 pneumonia caused by SARS-CoV-2.

BACKGROUND: The current outbreak of coronavirus disease 2019 (COVID-19) caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan, Hubei, China, spreads across national and international borders. METHODS: We prospectively collected medical records of 14 health care workers (HCWs) who were infected with SARS-CoV-2, in neurosurgery department of Wuhan Union Hospital, China. RESULTS: Among the 14 HCWs, 12 were conformed cases, the other 2 were suspected cases. Most of them were either exposed to the two index patients or infected coworkers, without knowing they were COVID-19 patients. There were 4 male and 10 female infected HCWs in this cohort, whose mean age was 36 years (SD, 6 years). The main symptoms included myalgia or fatigue (100%), fever (86%) and dry cough (71%). On admission, 79% of infected HCWs showed leucopenia and 43% lymphopenia. Reduced complement C3 could be seen in 57% of the infected HCWs and IL-6 was significantly elevated in 86% of them. The proportion of lymphocytes subsets, concentrations of immunoglobulins, complement C4, IL-2, IL-4, IL-10, TNF-α and IFN-γ were within normal range in these 14 infected HCWs. The most frequent findings on pulmonary computed tomographic images were bilateral multifocal ground-glass opacifications (86%). CONCLUSIONS: Human-to-human transmission of COVID-19 pneumonia has occurred among HCWs, and most of these infected HCWs with confirmed COVID-19 are mild cases. Our data suggest that in the epidemic area of COVID-19, stringent and urgent surveillance and infection-control measures should be implemented to protect doctors and nurses from COVID-19 infection.

Accumulated Clinical Experiences from Successful Treatment of 1377 Severe and Critically Ill COVID-19 Cases.

In late December 2019, COVID-19 was firstly recognized in Wuhan, China and spread rapidly to all of the provinces of China. The West Campus of Wuhan Union Hospital, the designated hospital to admit and treat the severe and critically ill COVID-19 cases, has treated a large number of such patients with great success and obtained lots of valuable experiences based on the Chinese guideline (V7.0). To standardize and share the treatment procedures of severe and critically ill cases, Wuhan Union Hospital has established a working group and formulated an operational recommendation, including the monitoring, early warning indicators, and several treatment principles for severe and critically ill cases. The treatment experiences may provide some constructive suggestions for treating the severe and critically ill COVID-19 cases all over the world.

Diarrhea Is Associated With Prolonged Symptoms and Viral Carriage in Corona Virus Disease 2019.

BACKGROUND & AIMS: We compared clinical, laboratory, radiological, and outcome features of patients with SARS-CoV-2 infection (COVID-19) with pneumonia, with vs without diarrhea. METHODS: We performed a retrospective, single-center analysis of 84 patients with SARS-CoV-2 pneumonia in Wuhan Union Hospital, China, from January 19 through February 7, 2020. Cases were confirmed by real-time reverse-transcriptase PCR of nasal and pharyngeal swab specimens for SARS-CoV-2 RNA. Blood samples were analyzed for white blood cell count, lymphocyte count, alanine aminotransferase, creatine kinase, lactate dehydrogenase, D-dimer, C-reactive protein, and in some cases, immunoglobulins, complement, lymphocyte subsets, and cytokines. Virus RNA was detected in stool samples by real-time PCR. RESULTS: Of the 84 patients with SARS-CoV-2 pneumonia, 26 (31%) had diarrhea. The duration of fever and dyspnea in patients with diarrhea was significantly longer than those without diarrhea (all P < .05). Stool samples from a higher proportion of patients with diarrhea tested positive for virus RNA (69%) than from patients without diarrhea (17%) (P < .001). As of February 19, a lower proportion of patients with diarrhea had a negative result from the latest throat swab for SARS-CoV-2 (77%) than patients without diarrhea (97%) (P = .010), during these patients' hospitalization. Of 76 patients with a negative result from their latest throat swab test during hospitalization, a significantly higher proportion of patients with diarrhea had a positive result from the retest for SARS-CoV-2 in stool (45%) than patients without diarrhea (20%) (P = .039). CONCLUSIONS: At a single center in Wuhan, China, 31% of patients with SARS-CoV-2 pneumonia had diarrhea. A significantly higher proportion of patients with diarrhea have virus RNA in stool than patients without diarrhea. Elimination of SARS-CoV-2 from stool takes longer than elimination from the nose and throat.

Interleukin-26 upregulates interleukin-22 production by human CD4+ T cells in tuberculous pleurisy.

IL-26 is a potentially important player in host defense and may be a pathogenic factor in the chronic inflammatory disorders of humans. However, the involvement of IL-26 in tuberculous pleural effusion (TPE) has not been investigated. The concentration of IL-26 was determined in pleural fluids and sera from patients with pleural effusions. Flow cytometry was performed to identify the cell origin of IL-26. The effects of tuberculosis-specific antigen (ESAT-6/CFP-10) on IL-26 expression of CD4+ T cell were explored. The impacts of IL-26 on modulating CD4+ T cell polarization were also investigated. The concentrations of IL-26 were much higher in tuberculous, malignant, and infectious PE than those in the corresponding serum. The expression of IL-26 on CD4+ T cells was much higher in tuberculous PE than those in the corresponding serum, and pleural Th1 and Th17 cells might be the major cell sources of IL-26. The addition of ESAT-6/CFP-10 to CD4+ T cells led to increasing the number of IL-26-producing CD4+ T cells and IL-26 expression on Th1 and Th17 cells. IL-26 could induce the differentiation and generation of IL-22 by memory and naive CD4+ T cells. IL-26 also upregulated the mRNA encoding CC-chemokine ligand 20 (CCL20) and CCL22 by mononuclear cells isolated from TPE. This study implies that pleural Th1 and Th17 cells are the major cell sources of IL-26, which could induce the differentiation and generation of Th22 cells by CD4+ T cells, suggesting the involvement of IL-26 in the pathogenesis of human TPE. KEY MESSAGES: IL-26 is overexpressed in TPE patients and presents a higher concentration in pleural effusion than the corresponding peripheral blood. Pleural Th1 and Th17 cells might be the major cell sources of IL-26 in TPE patients. IL-26 promotes IL-22 secretion and Th22 generation by CD4+ T cells isolated from TPE patients. IL-26 may play an active role in the pathogenesis of tuberculous pleurisy.

Diagnostic accuracy of interleukin 27 for tuberculous pleural effusion: two prospective studies and one meta-analysis.

BACKGROUND: Accurate differentiating diagnosis is essential for choosing treatment for exudative pleural effusions. OBJECTIVE: To establish the diagnostic accuracy of interleukin 27 for tuberculous pleural effusion (TPE). METHODS: First, the concentrations of pleural interleukin 27, interferon-gamma and adenosine deaminase were compared between 51 patients with TPE and 103 with non-TPEs (Beijing cohort), and their diagnostic values were evaluated. These were further verified in another independent population (Wuhan cohort, n=120). In the second part of the study, we performed a meta-analysis. RESULTS: With a cut-off value of 591.4 ng/L in the Beijing cohort, the area under the curve, sensitivity, specificity, positive predictive value and negative predictive value of interleukin 27 to diagnose TPE were 0.983 (95% CI 0.947 to 0.997), 96.1% (86.5% to 99.5%), 99.0% (94.7% to 100%), 98.0 (89.4 to 99.9) and 98.1 (93.3 to 99.8), respectively. Excellent diagnostic accuracy of interleukin 27 was also found in the Wuhan cohort and was further confirmed in the meta-analysis. The diagnostic performance of interleukin 27 was comparable to that of interferon-gamma and was more accurate than that of adenosine deaminase. Since the post-test probability of a negative result was always <0.1%, a negative test was considered to exclude TPE in all tuberculosis prevalence settings. CONCLUSIONS: Interleukin 27 can be used to diagnose TPE in a high prevalence setting, and a negative result can also be reliably used to rule out TPE in all prevalence settings.

Multiple pulmonary metastases with halo-sign from malignant mixed Müllerian tumors.

The lungs are one of the most common organs to which cancer metastasizes, but are a location not common for uterine sarcoma. A malignant mixed Müllerian tumor (MMMT) of the uterus is an extremely rare and aggressive sarcoma, characterized by a mixture of epithelial and mesenchymal components. There are few reports regarding the pulmonary metastasis from MMMTs. The present study presents the case of a 58-year-old woman with hemoptysis and post-menopausal vaginal bleeding. The woman was initially diagnosed with invasive aspergillosis based on a chest computed tomography (CT) scan showing multiple pulmonary nodular opacities surrounded by a ground-glass attenuation halo (halo-sign). Diagnostic curettage and a percutaneous CT-guided lung biopsy were conducted for the pathological diagnosis. Finally, the diagnosis was confirmed as MMMT with lung metastasis based on the histopathological examination of cervical canals, uterus and lung specimens, which showed a mixture of carcinomatous and sarcomatous elements, and morphology exhibiting hyperchromatic nuclei and necrosis. Immunohistochemical staining was positive for vimentin, focally positive for p16, and negative for napsin, cytokeratin 7 (CK7), CK20, carcinoembryonic antigen, carbohydrate antigen 125, homeobox protein CDX2 and villin in the lung specimens. This case highlights that pulmonary metastatic tumor from uterine sarcoma can present as halo-sign, which is commonly observed in pulmonary aspergillosis. Therefore, it needs to be considered in the differential diagnosis of such lesions, and pathological confirmation is required.

miR-18a-5p Inhibits Sub-pleural Pulmonary Fibrosis by Targeting TGF-ß Receptor II.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease that typically leads to respiratory failure and death within 3-5 years of diagnosis. Sub-pleural pulmonary fibrosis is a pathological hallmark of IPF. Bleomycin treatment of mice is a an established pulmonary fibrosis model. We recently showed that bleomycin-induced epithelial-mesenchymal transition (EMT) contributes to pleural mesothelial cell (PMC) migration and sub-pleural pulmonary fibrosis. MicroRNA (miRNA) expression has recently been implicated in the pathogenesis of IPF. However, changes in miRNA expression in PMCs and sub-pleural fibrosis have not been reported. Using cultured PMCs and a pulmonary fibrosis animal model, we found that miR-18a-5p was reduced in PMCs treated with bleomycin and that downregulation of miR-18a-5p contributed to EMT of PMCs. Furthermore, we determined that miR-18a-5p binds to the 3' UTR region of transforming growth factor ß receptor II (TGF-ßRII) mRNA, and this is associated with reduced TGF-ßRII expression and suppression of TGF-ß-Smad2/3 signaling. Overexpression of miR-18a-5p prevented bleomycin-induced EMT of PMC and inhibited bleomycin-induced sub-pleural fibrosis in mice. Taken together, our data indicate that downregulated miR-18a-5p mediates sub-pleural pulmonary fibrosis through upregulation of its target, TGF-ßRII, and that overexpression of miR-18a-5p might therefore provide a novel approach to the treatment of IPF.

Imbalance between subsets of CD8(+) peripheral blood T cells in patients with chronic obstructive pulmonary disease.

Background. CD8(+) T lymphocytes are known to play a critical role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, systematic analyses of CD8(+) T cell (Cytotoxic T cells, Tc) subsets in COPD patients have yet to be well conducted. Methods. The whole Tc subsets, including Tc1/2/10/17, CD8(+) regulatory T cells (Tregs) and CD8(+) α7(+) T cells, were quantified by flow cytometry in peripheral blood from 24 stable COPD subjects (SCOPD), 14 patients during acute exacerbations (AECOPD), and 14 healthy nonsmokers (HN). Results. Acute exacerbations of COPD were accompanied by elevated levels of circulating CD8(+) T cells. Tc1 cells were increased in both SCOPD and AECOPD patients, whereas the percentage of Tc2 cells was decreased in SCOPD patients but remained normal in AECOPD patients. Tc17 cells were increased only in AECOPD patients, and the percentage of Tc10 cells was reduced in both SCOPD and AECOPD patients. The imbalances of pro/anti-inflammatory Tc subsets observed in COPD may be caused by the lack of Tc10 cells and the impaired anti-inflammatory capacity of CD8(+) Tregs. Conclusions. The imbalances between subsets of CD8(+) peripheral blood T cells contribute to the immune response dysfunction in COPD pathogenesis.

TGF-ß/BAMBI pathway dysfunction contributes to peripheral Th17/Treg imbalance in chronic obstructive pulmonary disease.

BMP and activin membrane-bound inhibitor (BAMBI) is postulated to inhibit or modulate transforming growth factor ß (TGF-ß) signaling. Furthermore, strong upregulation of BAMBI expression following in vitro infection of chronic obstructive pulmonary disease (COPD) lung tissue has been demonstrated. In this study, we investigated whether TGF-ß/BAMBI pathway is associated with COPD. Blood samples were obtained from 27 healthy controls (HC), 24 healthy smokers (HS) and 29 COPD patients. Elevated Th17/Treg ratios, and increased levels of BAMBI protein and mRNA (in plasma and CD4(+) T cells respectively), were observed in COPD compared with HC and HS. BAMBI expression was first observed on human CD4(+) T cells, with a typical membrane-bound pattern. The enhanced plasma BAMBI levels in COPD positively correlated with the increased plasma TGF-ß1 levels and Th17/Treg ratio. Together, an impaired TGF-ß/BAMBI pathway may promote the inflammation leading to Th17/Treg imbalance, which is a new mechanism in smokers who develop COPD.

Cigarette Smoke Disturbs the Survival of CD8+ Tc/Tregs Partially through Muscarinic Receptors-Dependent Mechanisms in Chronic Obstructive Pulmonary Disease.

BACKGROUND: CD8+ T cells (Cytotoxic T cells, Tc) are known to play a critical role in the pathogenesis of smoking related airway inflammation including chronic obstructive pulmonary disease (COPD). However, how cigarette smoke directly impacts systematic CD8+ T cell and regulatory T cell (Treg) subsets, especially by modulating muscarinic acetylcholine receptors (MRs), has yet to be well elucidated. METHODS: Circulating CD8+ Tc/Tregs in healthy nonsmokers (n = 15), healthy smokers (n = 15) and COPD patients (n = 18) were evaluated by flow cytometry after incubating with anti-CD3, anti-CD8, anti-CD25, anti-Foxp3 antibodies. Peripheral blood T cells (PBT cells) from healthy nonsmokers were cultured in the presence of cigarette smoke extract (CSE) alone or combined with MRs agonist/antagonist for 5 days. Proliferation and apoptosis were evaluated by flow cytometry using Ki-67/Annexin-V antibodies to measure the effects of CSE on the survival of CD8+ Tc/Tregs. RESULTS: While COPD patients have elevated circulating percentage of CD8+ T cells, healthy smokers have higher frequency of CD8+ Tregs. Elevated percentages of CD8+ T cells correlated inversely with declined FEV1 in COPD. CSE promoted the proliferation and inhibited the apoptosis of CD8+ T cells, while facilitated both the proliferation and apoptosis of CD8+ Tregs. Notably, the effects of CSE on CD8+ Tc/Tregs can be mostly simulated or attenuated by muscarine and atropine, the MR agonist and antagonist, respectively. However, neither muscarine nor atropine influenced the apoptosis of CD8+ Tregs. CONCLUSION: The results imply that cigarette smoking likely facilitates a proinflammatory state in smokers, which is partially mediated by MR dysfunction. The MR antagonist may be a beneficial drug candidate for cigarette smoke-induced chronic airway inflammation.

Immune Regulation of Interleukin-27 in Malignant Pleural Effusion.

BACKGROUND: Interleukin (IL)-27 has been reported to have anti-proliferate and anti-angiogenic activities on cancer cells. However, the involvement of IL-27 in malignant pleural effusion (MPE) remains unknown. Thus, in this research, we compared the immune functions of IL-27, interferon (IFN)-γ, and IL-17 on lung cancer cells and revealed the regulatory mechanism of IL-27 in MPE. METHODS: The distribution of IL-27 in both MPE and blood was evaluated by enzyme-linked immunosorbent assay and flow cytometry. The expressions of cytokine receptors and the levels of the phosphorylated signal transducer and activator of transcription (STAT) signalings were detected by flow cytometry. As well as the effects of proliferation, apoptosis, migration, and adherent activity of IL-27, IFN-γ, and IL-17 on lung cancer cells were also explored. RESULTS: The expression of IL-27 was increased in MPE when compared with blood (147.3 ± 25.1 pg/ml vs. 100.3 ± 13.9 pg/ml, P = 0.04). IL-27 was noted to suppress both proliferation (18.33 ± 0.21 vs. 27.77 ± 0.88, P = 0.0005) and migration (1.82 ± 0.44 vs. 3.13 ± 0.07, P = 0.04) of A549 cells, but obviously promoted apoptosis of A549 cells (9.47 ± 1.14 vs. 4.96 ± 0.17, P = 0.02) by activating STAT1 signaling. Interestingly, IL-27 played totally opposite effects on A549 cells by activating STAT3 pathway. Moreover, IL-27 exerted different intercellular adherent activities of A549 cells to pleural mesothelial cell monolayer by activating different STAT signalings. CONCLUSIONS: IL-27 might exert an important immune regulation on lung cancer cells in human pleural malignant environment.

Bleomycin induced epithelial-mesenchymal transition (EMT) in pleural mesothelial cells.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis. Recent studies revealed that pleural mesothelial cells (PMCs) undergo epithelial-mesenchymal transition (EMT) and play a pivotal role in IPF. In animal model, bleomycin induces pulmonary fibrosis exhibiting subpleural fibrosis similar to what is seen in human IPF. It is not known yet whether bleomycin induces EMT in PMCs. In the present study, PMCs were cultured and treated with bleomycin. The protein levels of collagen-I, mesenchymal phenotypic markers (vimentin and α-smooth muscle actin), and epithelial phenotypic markers (cytokeratin-8 and E-cadherin) were measured by Western blot. PMC migration was evaluated using wound-healing assay of culture PMCs in vitro, and in vivo by monitoring the localization of PMC marker, calretinin, in the lung sections of bleomycin-induced lung fibrosis. The results showed that bleomycin induced increases in collagen-I synthesis in PMC. Bleomycin induced significant increases in mesenchymal phenotypic markers and decreases in epithelial phenotypic markers in PMC, and promoted PMC migration in vitro and in vivo. Moreover, TGF-ß1-Smad2/3 signaling pathway involved in the EMT of PMC was demonstrated. Taken together, our results indicate that bleomycin induces characteristic changes of EMT in PMC and the latter contributes to subpleural fibrosis.

Cigarette smoking promotes inflammation in patients with COPD by affecting the polarization and survival of Th/Tregs through up-regulation of muscarinic receptor 3 and 5 expression.

BACKGROUND: CD4+ T cells in the lung are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), although CD4+ T cell subsets and the direct effect of smoking on these cells, especially the expression of MRs, have not been comprehensively examined. METHODS: First, circulating CD4+ T cell subsets in healthy nonsmokers, patients with SCOPD and patients with AECOPD were evaluated by flow cytometry. Then, differentiation experiments were carried out using RT-PCR, and Ki-67/Annexin V antibodies were used to measure proliferation and apoptosis. We also explored the impact of CSE on the differentiation and survival of CD4+Th/Tregs and examined the expression of MRs in healthy nonsmokers and patients with SCOPD. RESULTS: We found the percentages of circulating Th1 and Th17 cells were increased in patients with AECOPD, while the percentage of Th2 cells was decreased in patients with SCOPD. The percentages of Th10 cells were decreased in both patients with SCOPD and patients with AECOPD, while the percentages of Tregs were increased. In addition, the percentages of CD4+α-7+ T cells were decreased in patients with SCOPD and patients with AECOPD. However, only the decrease observed in patients with AECOPD was significant. In vitro studies also revealed MR expression affected the polarization of T cells, with different CD4+ T cell subtypes acquiring different MR expression profiles. The addition of CSE facilitated CD4+ T cell polarization towards pro-inflammatory subsets (Th1 and Th17) and affected the survival of CD4+ T cells and Treg cells by up-regulating the expression of MR3 and 5, resulting in an imbalance of CD4+ T cell subsets. CONCLUSIONS: Our findings suggest an imbalance of circulating CD4+ T cell subsets is involved in COPD pathogenesis in smokers. Cigarette smoking may contribute to this imbalance by affecting the polarization and survival of Th/Tregs through the up-regulation of MR3 and MR5.

Treg/IL-17 ratio and Treg differentiation in patients with COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by chronic pulmonary and systematic inflammation. An abnormal adaptive immune response leads to an imbalance between pro- and anti-inflammatory processes. T-helper (Th), T-cytotoxic (Tc) and T-regulatory (Treg) cells may play important roles in immune and inflammatory responses. This study was conducted to clarify the changes and imbalance of cytokines and T lymphocyte subsets in patients with COPD, especially during acute exacerbations (AECOPD). METHODS: Twenty-three patients with stable COPD (SCOPD) and 21 patients with AECOPD were enrolled in the present study. In addition, 20 age-, sex- and weight-matched non-smoking healthy volunteers were included as controls. The serum levels of selected cytokines (TGF-ß, IL-10, TNF-α, IL-17 and IL-9) were measured by enzyme-linked immunosorbent assay (ELISA) kits. Furthermore, the T lymphocyte subsets collected from peripheral blood samples were evaluated by flow cytometry after staining with anti-CD3-APC, anti-CD4-PerCP, anti-CD8- PerCP, anti-CD25-FITC and anti-FoxP3-PE monoclonal antibodies. Importantly, to remove the confounding effects of inflammatory factors, the authors introduced a concept of "inflammation adjustment" and corrected each measured value using representative inflammatory markers, such as TNF-α and IL-17. RESULTS: Unlike the other cytokines, serum TGF-ß levels were considerably higher in patients with AECOPD relative to the control group regardless of adjustment. There were no significant differences in the percentages of either CD4+ or CD8+ T cells among the three groups. Although Tregs were relatively upregulated during acute exacerbations, their capacities of generation and differentiation were far from sufficient. Finally, the authors noted that the ratios of Treg/IL-17 were similar among groups. CONCLUSIONS: These observations suggest that in patients with COPD, especially during acute exacerbations, both pro-inflammatory and anti-inflammatory reactions are strengthened, with the pro-inflammatory reactions dominating. Although the Treg/IL-17 ratios were normal, the regulatory T cells were still insufficient to suppress the accompanying increases in inflammation. All of these changes suggest a complicated mechanism of pro- and anti-inflammatory imbalance which needs to be further investigated.

IL-33 levels differentiate tuberculous pleurisy from malignant pleural effusions.

Tuberculous pleural effusions (TPEs) and malignant pleural effusions (MPEs) are difficult to differentiate between in certain clinical situations. Interleukin (IL)-33 is a cytokine that participates in inflammatory responses and may have a role in pleural effusions. The present study aimed to investigate the concentrations and potential differential significance of IL-33 in patients with TPE and MPE. IL-33 levels in pleural effusion and serum samples were detected using sandwich enzyme-linked immunosorbent assay in 23 patients with TPE and 21 patients with MPE. The concentration of IL-33 (mean ± standard deviation) in the TPE patients (22.962±0.976 ng/l) was significantly higher than that in the MPE patients (12.603±5.153 ng/l; P<0.001; z=-4.572); however, there was no significant difference in the serum level of IL-33 in the patients with TPE compared with those with MPE (P>0.05). The concentration of IL-33 in the pleural effusions was positively correlated with that in the serum samples in each group (TPE: r=0.563, P=0.05; MPE: r=0.535, P<0.05). The cut-off value of pleural IL-33 for TPE was 19.86 ng/l, which yielded a sensitivity of 0.869, a specificity of 0.905 and an area under the corresponding receiver operating characteristic curve of 0.903. The present study identified that the level of pleural IL-33 is significantly increased in TPEs and may serve as a novel biomarker to differentiate between patients with TPE and MPE.