Simultaneous online SPE-HPLC-MS/MS quantification of gefitinib, osimertinib and icotinib in dried plasma spots: Application to therapeutic drug monitoring in patients with non-small cell lung cancer.

Gefitinib, osimertinib and icotinib are the most commonly used tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) with EGFR mutation. Therapeutic drug monitoring (TDM) for these TKIs has become a standard and essential procedure. Dried plasma spots (DPS) was choosen for microsampling strategies for TDM, allowing easy and cost-effective logistics in many settings. This study developd and validated an assay for the simultaneous quantitative determination of gefitinib, osimertinib and icotinib in DPS by online solid-phase extraction-liquid chromatography-tandem mass spectrometry (online SPE-LC-MS) system. The TKIs were extracted from DPS with methanol and enriched on a Welch Polar-RP SPE column (30 × 4.6 mm, 5 µm), followed by separation on Waters X Bridge C18 analytical column(4.6 × 100 mm, 3.5 µm). The method achieved LLOQ of 2 ng mL-1 for gefitinib and osimertinib (4 ng mL-1 for icotinib), respectively (r2 > 0.99). Precision (within-run 1.54-7.41 % RSD; between-run 3.03-12.84 % RSD), accuracy (range from 81.47 % to 105.08 %; between-run bias 87.87-104.13 %). Osimertinib and icotinib were stable in DPS stored at - 40 °C for 30 days, 4 °C, 42 °C and 60 °C for 5 days and well-sealed 37 °C,75 % humidity (except gefitinib). Lastly, the assay was applied to TDM of TKIs in 46 patients and the results were compared to SALLE assisted LC-MS analysis, it could be confirmed that the developed method achieves similarly good results as the already established one and no bias could be detected. It implies that this method capable of supporting clinical follow-up TDM of TKIs in DPS from poor medical environment.

Development and validation of a simple tool composed of items on dyspnea, respiration rates, and C-reactive protein for pneumonia prediction among acute febrile respiratory illness patients in primary care settings.

BACKGROUND: Acute febrile respiratory illness (AFRI) patients are susceptible to pneumonia and suffer from significant morbidity and mortality throughout the world. In primary care settings, the situation is worse. Limited by computerized tomography resources and physician experiences, AFRI patients in primary care settings may not be diagnosed appropriately, which would affect following treatment. In this study, we aimed to develop and validate a simple prediction model to help physicians quickly identify AFRI patients of pneumonia risk in primary care settings. METHODS: A total of 1977 AFRI patients were enrolled at two fever clinics in Shanghai, China, and among them, 727 patients who underwent CT scans were included in the analysis. Acute alveolar or interstitial infiltrates found on CT images were diagnosed with pneumonia. Characteristics and blood parameters were compared between pneumonia and non-pneumonia patients. Then a multivariable model for pneumonia prediction was developed through logistic regression analysis. Its value for pneumonia prediction was prospectively assessed in an external multi-center population, which included 1299 AFRI patients in primary settings from 5 different provinces throughout China. RESULTS: In the model development population, pneumonia patients (n = 227) had a longer duration of fever; higher frequencies of purulent sputum, dyspnea, and thoracic pain; and higher levels of respiration rates and C-reactive protein (CRP) than non-pneumonia patients (n = 500). Logistic regression analysis worked out a model composed of items on dyspnea, respiration rates > 20/min, and CRP > 20 mg/l (DRC) for pneumonia prediction with an area under curve (AUC) of 0.8506. In the external validation population, the predictive accuracy of the DRC model was the highest when choosing at least one positive item (1 score) as a cut-off point with a sensitivity of 87.0% and specificity of 80.5%. DRC scores increased with pneumonia severity and lung lobe involvement and showed good performance for both bacterial and viral pneumonia. For viral pneumonia, dyspnea plus respiration rates > 20/min had good predictive capacity regardless of CRP concentration. CONCLUSIONS: DRC model is a simple tool that predicts pneumonia among AFRI patients, which would help physicians utilize medical resources rationally in primary care settings.

Multidrug-resistant Pseudomonas aeruginosa is predisposed to lasR mutation through up-regulated activity of efflux pumps in non-cystic fibrosis bronchiectasis patients.

Background: Multidrug-resistant (MDR) Pseudomonas aeruginosa is a frequent opportunistic pathogen that causes significant mortality in patients with non-cystic fibrosis bronchiectasis (NCFB). Although the quorum sensing (QS) system is a potential target for treatment, lasR mutants that present with a QS-deficient phenotype have been frequently reported among clinical P. aeruginosa isolates. We aimed to investigate whether antibiotic resistance would select for lasR mutants during chronic P. aeruginosa lung infection and determine the mechanism underlying the phenomenon. Methods: We prospectively evaluated episodes of chronic P. aeruginosa lung infections in NCFB patients over a 2-year period at two centers of our institution. QS phenotypic assessments and whole-genome sequencing (WGS) of P. aeruginosa isolates were performed. Evolution experiments were conducted to confirm the emergence of lasR mutants in clinical MDR P. aeruginosa cultures. Results: We analyzed episodes of P. aeruginosa infection among 97 NCFB patients and found only prior carbapenem exposure independently predictive of the isolation of MDR P. aeruginosa strains. Compared with non-MDR isolates, MDR isolates presented significantly QS-deficient phenotypes, which could not be complemented by the exogenous addition of 3OC12-HSL. The paired isolates showed that their QS-phenotype deficiency occurred after MDR was developed. Whole-genome sequencing analysis revealed that lasR nonsynonymous mutations were significantly more frequent in MDR isolates, and positive correlations of mutation frequencies were observed between genes of lasR and negative-efflux-pump regulators (nalC and mexZ). The addition of the efflux pump inhibitor PAßN could not only promote QS phenotypes of these MDR isolates but also delay the early emergence of lasR mutants in evolution experiments. Conclusions: Our data indicated that MDR P. aeruginosa was predisposed to lasR mutation through the upregulated activity of efflux pumps. These findings suggest that anti-QS therapy combined with efflux pump inhibitors might be a potential strategy for NCFB patients in the challenge of MDR P. aeruginosa infections.

Tracking the response to Pseudomonas aeruginosa infection in ozone-induced chronic obstructive pulmonary disease mouse models.

Pseudomonas aeruginosa (P. aeruginosa) is commonly isolated from the sputum of COPD patients. However, the precise role of P. aeruginosa infection in the progression of COPD, especially its role in altering inflammation remains unclear. Here, we designed mice models of COPD infected with P. aeruginosa (PA) and observed dynamic changes of lung structure, lung inflammatory microenvironment, lung function. After infection, the level of mucus secretion peaked on day 3 and remained higher throughout the study period, and the airway remodeling and emphysema was starkly apparent on day 14 and 21. On day 3, interferon-γ and interleukin (IL)- 5 levels increased rapidly, accompanied by elevated T-bet mRNA expression and CD4+T-bet+ cells; at the late stage of infection (days 14 and 21), consistent with increased GATA3 mRNA expression and CD4+GATA3+ cells, IL-4 and IL-13 levels significantly increased; IL-17A level, Foxp3 mRNA expression, CD4+ROR-γt+ cells and CD4+FOXP3+ cells remained at higher levels throughout the course of the infection. Small-airway function showed a decline from day 3 to day 21; large airway function showed a decline on day 14 and 21. Overall, P. aeruginosa infection contributed to the progression of COPD. During the infection, an early Th1-related inflammation gradually shifted to a later Th2-related inflammation, and small-airway function decline occurred earlier than that of large-airway function. On the basis of infection control, the appropriate use of glucocorticoid might slow disease progression by mitigating the enhanced Th2-related inflammation, and small airways could be also an important treatment target in P. aeruginosa -infected COPD patients.

Early Fever Is Associated With Clinical Outcomes in Patients With Coronavirus Disease.

Fever is one of the typical symptoms of coronavirus disease (COVID-19). We aimed to investigate the association between early fever (EF) and clinical outcomes in COVID-19 patients. A total of 1,014 COVID-19 patients at the Leishenshan Hospital were enrolled and classified into the EF and non-EF groups based on whether they had fever within 5 days of symptom onset. Risk factors for clinical outcomes in patients with different levels of disease severity were analyzed using multivariable analyses. Time from symptom onset to symptom alleviation, CT image improvement, and discharge were longer for patients with moderate and severe disease in the EF group than in the non-EF group. Multivariable analysis showed that sex, EF, eosinophil number, C-reactive protein, and IL-6 levels were positively correlated with the time from symptom onset to hospital discharge in moderate cases. The EF patients showed no significant differences in the development of acute respiratory distress syndrome, compared with the non-EF patients. The Kaplan-Meier curve showed no obvious differences in survival between the EF and non-EF patients. However, EF patients with increased temperature showed markedly lower survival than the non-EF patients with increased temperature. EF had no significant impact on the survival of critically ill patients, while an increase in temperature was identified as an independent risk factor. EF appears to be a predictor of longer recovery time in moderate/severe COVID-19 infections. However, its value in predicting mortality needs to be considered for critically ill patients with EF showing increasing temperature.

(-)-Epicatechin ameliorates cigarette smoke-induced lung inflammation via inhibiting ROS/NLRP3 inflammasome pathway in rats with COPD.

Chronic obstructive pulmonary disease (COPD) with increased morbidity and mortality is a worldwide healthcare challenge closely associated with cigarette smoking (CS). Currently, there is no effective therapeutic strategy to control inflammation in COPD patients. The present study tested the protective effects of (-)-Epicatechin (EC), a type of flavonoid, on CS-induced COPD and the underlying mechanism. Also, EC repressed the production of reactive oxygen species (ROS) and improved human bronchial epithelial cell viability after cigarette smoke extract (CSE) treatment. Further studies demonstrated that EC promotes ubiquitin-mediated Keap1 degradation by upregulating tripartite motif-containing protein 25 (TRIM25) expression and enhances the nuclear localization of Nrf2 protein. Also, EC dramatically inhibits the activation of NLRP3 inflammasome and reduces the CSE-induced pyroptosis, as indicated by decreasing lactate dehydrogenase release and the number of caspase-1-positive cells. Importantly, Nrf2 knockdown reversed the protective effect of EC on human bronchial epithelial cells, at least partially. Consistent with the results in vitro, EC inhibits the activation of NLRP3 inflammasome and relieves the CS-induced lung inflammation, as evident from decreased interleukin (IL)-1ß and IL-18 secretion in a COPD rat model. In conclusion, this study revealed the protective effect of EC on experimental COPD rats and elucidated the mechanism of EC promoting Nrf2 activity, which might provide a novel therapeutic strategy for COPD.

Clinical features in coronavirus disease 2019 (COVID-19) patients with early clearance and prolonged shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA.

BACKGROUND: Since the outbreak of coronavirus disease 2019 (COVID-19), the pattern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA shedding has not been well characterized. METHODS: In our study, 652 patients in Wuhan Designated Hospital were recruited, and their clinical and laboratory findings were extracted and analyzed. RESULTS: The median duration of SARS-CoV-2 RNA detection was 23 days [interquartile range (IQR), 18 days] from symptom onset. Compared to patients with early viral RNA clearance (<23 days after illness onset), we found that patients with late viral RNA clearance (≥23 days) had a higher proportion of clinical features, as follows: symptoms, including fever, dry cough, and sputum production; comorbidities, including hypertension, chronic kidney disease, uremia, chronic liver disease, anemia, hyperlipidemia, and bilateral lung involvement; complications, such as liver injury; delayed admission to hospital; laboratory parameters at baseline, including higher eosinophils, uric acid, cholesterol, triglycerides, and lower hemoglobin; and less treatment with arbidol, chloroquine, or any antivirals. After generalized linear regression, prolonged SARS-CoV-2 RNA shedding was independently associated with younger age; delayed admission to hospital; symptoms including fever, shivering, and sputum production; comorbidities including hypertension, diabetes, cardiovascular disease, anemia, hyperlipidemia, uremia, and lung involvement; and higher alanine aminotransferase (ALT), uric acid, and cholesterol levels at baseline. CONCLUSIONS: In conclusion, the factors mentioned above are associated with the negative conversion of SARS-CoV-2 RNA. A deeper insight into virological dynamics will be helpful for establishing patient discharge and quarantine release criteria.

The role of peripheral blood eosinophil counts in COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) emerged in Wuhan city and rapidly spread globally outside China. We aimed to investigate the role of peripheral blood eosinophil (EOS) as a marker in the course of the virus infection to improve the efficiency of diagnosis and evaluation of COVID-19 patients. METHODS: 227 pneumonia patients who visited the fever clinics in Shanghai General Hospital and 97 hospitalized COVID-19 patients admitted to Shanghai Public Health Clinical Center were involved in a retrospective research study. Clinical, laboratory, and radiologic data were collected. The trend of EOS level in COVID-19 patients and comparison among patients with different severity were summarized. RESULTS: The majority of COVID-19 patients (71.7%) had a decrease in circulating EOS counts, which was significantly more frequent than other types of pneumonia patients. EOS counts had good value for COVID-19 prediction, even higher when combined with neutrophil-to-lymphocyte ratio. Patients with low EOS counts at admission were more likely to have fever, fatigue, and shortness of breath, with more lesions in chest CT and radiographic aggravation, and longer length of hospital stay and course of disease than those with normal EOS counts. Circulating EOS level gradually increased over the time, and was synchronous with the improvement in chest CT (12 days vs 13 days, P = .07), later than that of body temperature (12 days vs 10 days, P = .014), but earlier than that of the negative conversion of nucleic acid assays (12 days vs 17 days, P = .001). CONCLUSION: Peripheral blood EOS counts may be an effective and efficient indicator in diagnosis, Evaluation, and prognosis monitoring of COVID-19 patients.

IL-17 Aggravates Pseudomonas aeruginosa Airway Infection in Acute Exacerbations of Chronic Obstructive Pulmonary Disease.

Pseudomonas aeruginosa airway infection increases risks of exacerbations and mortality in chronic obstructive pulmonary disease (COPD). We aimed to elucidate the role of IL-17 in the pathogenesis. We examined the expression and influences of IL-23/IL-17A in patients with stable COPD (n = 33) or acute COPD exacerbations with P. aeruginosa infection (n = 34). A mouse model of COPD (C57BL/6) was used to investigate the role of IL-17A in host inflammatory responses against P. aeruginosa infection through the application of IL-17A-neutralizing antibody or recombinant IL-17A. We found that P. aeruginosa infection increased IL-23/17A signaling in lungs of both COPD patients and COPD mouse models. When COPD mouse models were treated with neutralizing antibody targeting IL-17A, P. aeruginosa induced a significantly less polymorphonuclear leukocyte infiltration and less bacterial burden in their lungs compared to those of untreated counterparts. The lung function was also improved by neutralizing antibody. Furthermore, IL-17A-signaling blockade significantly reduced the expression of pro-inflammatory cytokine IL-1ß, IL-18, TNF-α, CXCL1, CXCL15 and MMP-9, and increased the expression of anti-inflammatory cytokine IL-10 and IL-1Ra. The application of mouse recombinant IL-17A exacerbated P. aeruginosa-mediated inflammatory responses and pulmonary dysfunction in COPD mouse models. A cytokine protein array revealed that the expression of retinol binding protein 4 (RBP4) was down-regulated by IL-17A, and exogenous RBP4-recombinant protein resulted in a decrease in the severity of P. aeruginosa-induced airway dysfunction. Concurrent application of IL-17A-neutralizing antibody and ciprofloxacin attenuated airway inflammation and ventilation after inoculation of P. aeruginosa in COPD mouse models. Our results revealed that IL-17 plays a detrimental role in the pathogenesis of P. aeruginosa airway infection during acute exacerbations of COPD. Targeting IL-17A is a potential therapeutic strategy in controlling the outcomes of P. aeruginosa infection in COPD patients.

CXCR4 knockdown prevents inflammatory cytokine expression in macrophages by suppressing activation of MAPK and NF-κB signaling pathways.

BACKGROUND: Recent evidence has shown that C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in acute lung injury (ALI). Macrophages are key factors in the pathogenesis of ALI. The aim of this study was to investigate the role of CXCR4 in macrophages after lipopolysaccharide (LPS) stimulation and confirm that CXCR4 knockdown can inhibit inflammatory cytokines by suppressing mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathway activation. RESULTS: In this study, we found that CXCR4 expression in lung tissue of ALI was significantly increased using immunofluorescence. We also found that the expression of CXCR4 in macrophages sorted from bronchoalveolar lavage fluid (BALF) of ALI was obviously upregulated through RT-qPCR. After CXCR4 knockdown using siRNA, we found that the expression of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) was obviously down regulated in macrophages. Additionally, the phosphorylation of p38, Erk, and p65 was significantly decreased after CXCR4 knockdown through western blotting. CONCLUSIONS: Taken together, the present study suggests that CXCR4 knockdown may inhibit inflammatory cytokine expression in macrophages by suppressing MAPK and NF-κB signaling pathway activation. Therefore, CXCR4 knockdown may have potential clinical value in treating ALI.

LPS-induced MMP-9 expression is mediated through the MAPKs-AP-1 dependent mechanism in BEAS-2B and U937 cells.

AIM OF THE STUDY: Matrix metalloproteinases (MMPs) play a critical role in chronic obstructive pulmonary disease (COPD). This study investigated the role of mitogen-activated protein kinases (MAPKs) in MMP-9 secretion of BEAS-2B cells, a human bronchial epithelial cell line and U937 cells, a human myeloid leukaemia cell line, which could differentiate into macrophage, after LPS stimulation, and some details of involved signaling. MATERIALS AND METHODS: MTT assay was used to measure cell viability. U937 cells were incubated for 48h with 100ng/ml PMA, and had a resting period of 24h with culture medium without PMA for differentiation of U937 cells into macrophages. For the experiments, U937 cells or BEAS-2B cells were pretreated with several inhibitors and then stimulated by LPS. Western blotting, quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA) and DNA binding activity assay were used for measuring the protein expression, RNA expression, cytokine production and DNA binding activity, respectively. RESULTS: We found LPS induced MMP-9 expression and secretion were completely blocked by stress-activated protein kinase/jun kinase (SAPK/JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors but not by p38 inhibitor. LPS-induced transactivation of AP-1 was also inhibited by JNK inhibitor SP600125 and ERK1/2 inhibitor PD98059. CONCLUSIONS: The present study suggests that in BEAS-2B cells and U937 cells, LPS probably activates ERK1/2 pathway and JNK pathway, which in turn initiate AP-1 activity, and leading to MMP-9 expression. Thus the ERK1/2 inhibitor and JNK inhibitor may have potential clinical value in treating COPD.

The Pseudomonas aeruginosa Orphan Quorum Sensing Signal Receptor QscR Regulates Global Quorum Sensing Gene Expression by Activating a Single Linked Operon.

Pseudomonas aeruginosa uses two acyl-homoserine lactone signals and two quorum sensing (QS) transcription factors, LasR and RhlR, to activate dozens of genes. LasR responds to N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and RhlR to N-butanoyl-homoserine lactone (C4-HSL). There is a third P. aeruginosa acyl-homoserine-lactone-responsive transcription factor, QscR, which acts to dampen or delay activation of genes by LasR and RhlR by an unknown mechanism. To better understand the role of QscR in P. aeruginosa QS, we performed a chromatin immunoprecipitation analysis, which showed this transcription factor bound the promoter of only a single operon of three genes linked to qscR, PA1895 to PA1897. Other genes that appear to be regulated by QscR in transcriptome studies were not direct targets of QscR. Deletion of PA1897 recapitulates the early QS activation phenotype of a QscR-null mutant, and the phenotype of a QscR-null mutant was complemented by PA1895-1897 but not by PA1897 alone. We conclude that QscR acts to modulate quorum sensing through regulation of a single operon, apparently raising the QS threshold of the population and providing a "brake" on QS autoinduction.IMPORTANCE Quorum sensing, a cell-cell communication system, is broadly distributed among bacteria and is commonly used to regulate the production of shared products. An important consequence of quorum sensing is a delay in production of certain products until the population density is high. The bacterium Pseudomonas aeruginosa has a particularly complicated quorum sensing system involving multiple signals and receptors. One of these receptors, QscR, downregulates gene expression, unlike the other receptors in P. aeruginosa QscR does so by inducing the expression of a single operon whose function provides an element of resistance to a population reaching a quorum. This finding has importance for design of quorum sensing inhibitory strategies and can also inform design of synthetic biological circuits that use quorum sensing receptors to regulate gene expression.

Lentivirus-mediated overexpression of suppressor of cytokine signaling-3 reduces neutrophilic airway inflammation by suppressing T-helper 17 responses in mice with chronic Pseudomonas aeruginosa lung infections.

The aim of the present study was to explore the effect of overexpressed suppressor of cytokine signaling­3 (SOCS3) on T-helper (Th)17 cell responses and neutrophilic airway inflammation in mice with chronic Pseudomonas aeruginosa (PA) infections. SOCS3 expression was enhanced via the administration of tail vein injections of therapeutic lentivirus in mice with chronic PA lung infections. SOCS3 expression in the blood and lung tissue was assessed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis. Total and differential cell numbers and myeloperoxidase levels in the bronchoalveolar lavage (BAL) fluid were assessed, as well as the number of bacterial colonies in the lungs. Histological analysis of lung tissue was performed using hematoxylin and eosin staining and phosphorylated­signal transducer and activator of transcription­3 (p­STAT3) expression was measured by western blot analysis and immunohistochemistry. The expression of STAT3 mRNA and retinoid­related orphan receptor (ROR)γt were measured by RT­qPCR. The percentage of interleukin (IL)­17+ cells among cluster of differentiation (CD)4+ cells was calculated using flow cytometry and levels of IL­17A and IL­6 were assessed by ELISA. The expression of SOCS3 was significantly increased in CD4+ T cells following lentivirus injection and the inflammation of neutrophilic airways was notably ameliorated. Enhanced SOCS3 expression was associated with a significant decrease in the expression of p­STAT3 and RORγt in CD4+ T cells. Additionally, the percentage of IL­17+ cells among CD4+ T cells and the IL­17 contents in the BAL fluid were significantly decreased. Lentivirus­mediated overexpression of SOCS3 was revealed to ameliorate neutrophilic airway inflammation by inhibiting pulmonary Th17 responses in mice with chronic PA lung infections.

Upregulation of SOCS3 in lung CD4+ T cells in a mouse model of chronic PA lung infection and suppression of Th17­mediated neutrophil recruitment in exogenous SOCS3 transfer in vitro.

Neutrophilic airway inflammation in chronic lung infections caused by Pseudomonas aeruginosa (PA) is associated with T helper (Th)17 responses. Suppressor of cytokine signaling 3 (SOCS3) is the major negative modulator of Th17 function through the suppression of signal transducer and activator of transcription (STAT)3 activation. The aim of the present study was to investigate the expression of SOCS3 in lung CD4+ T cells in a mouse model of chronic PA lung infection and the effect of exogenous SOCS3 on Th17­mediated neutrophil recruitment in vitro. A mouse model of chronic PA lung infection was established and the activation of STAT3 and Th17 response in lung tissues and lung CD4+ T cells was assessed. The protein and mRNA expression of SOCS3 in lung CD4+ T cells was analyzed by western blotting and reverse transcription­quantitative polymerase chain reaction. The authors constructed a recombinant lentivirus carrying the SOCS3 gene and transferred it into lung CD4+ T cells isolated from a mouse model. These transfected cells were stimulated with interleukin (IL)­23 in vitro and the protein level of p­STAT3 and retinoid­related orphan receptor (ROR)γt was determined by western blotting. The expression of IL­17A+ cells was analyzed by flow cytometry and the level of IL­17A in cell culture supernatant was measured by ELISA. The mouse lung epithelial cell line, MLE­12, was cocultured with lung CD4+ T cells that overexpressed the SOCS3 gene and the culture supernatant was harvested and used for a chemotaxis assay. Compared with control mice, mice with chronic PA lung infection had significantly higher level of p­STAT3 and Th17 response in both lung tissues and lung CD4+ T cells. The protein and mRNA level of SOCS3 in lung CD4+ T cells increased as the chronic PA lung infection developed. Exogenous SOCS3 gene transfer in PA­infected lung CD4+ T cells decreased p­STAT3 and RORγt expression and suppressed the level of IL­17A+ cells in vitro. MLE­12 cells cocultured with SOCS3­overexpressing lung CD4+ T cells expressed a significantly lower level of neutrophil chemoattractants chemokine (C­X­C motif) ligand (CXCL) 1 and CXCL5, and recruited significantly smaller numbers of migrating neutrophils than those cocultured with control cells. SOCS3 was upregulated in lung CD4+ T cells following the activation of STAT3/Th17 axis in a mouse model of chronic PA lung infection. Exogenous SOCS3 transfer in PA­infected lung CD4+ T cells suppresses Th17­mediated neutrophil recruitment in vitro.

Plasma follistatin-like protein 1 is correlated with disease severity in patients with acute pulmonary embolism and predicts short-term mortality.

We investigated the plasma levels of follistatin-like protein 1 (FSTL1) in patients with acute pulmonary embolism (PE) and whether it could predict short-term mortality. A prospective observational cohort study was conducted in patients with acute PE (n = 220). FSTL1 was measured in plasma samples using enzyme-linked immunosorbent assay. Plasma FSTL1 levels were significantly increased in patients with acute PE, and positively correlated with disease severity (rs = 0.7171). Sensitivity and specificity rates for high-risk PE at a specific FSTL1 cutoff point (23 ng/ml) were 79.3% and 92.9%. Multivariant Cox regression analysis showed that FSTL1 was independently associated with 30-day mortality rate. Addition of FSTL1 to the PE severity index (PESI) scoring system significantly improved the predictive value for 30-day mortality. These results indicate that the plasma level of FSTL1 is correlated with disease severity in patients with acute PE and predicts short-term mortality.

Regulatory T cell activity is partly inhibited in a mouse model of chronic Pseudomonas aeruginosa lung infection.

OBJECTIVES: We aimed to investigate the activity of regulatory T (Treg) cells in chronic Pseudomonas aeruginosa (PA) lung infection and its influence on effector T-cell responses. MATERIALS AND METHODS: C57BL/6 mice were randomly inoculated with PA-laden agarose beads (1 × 10(5) CFU/50 µL) or planktonic PA (1 × 10(5) CFU/50 µL), and euthanized at the time points of 4 hour, day 1, 3, 5, and 7. Bacterial load, bronchoalveolar lavage (BAL) fluid cell counts, and lung tissue histology were assessed. BAL fluid concentrations of TGF-ß1, IFN-γ, IL-1ß, IL-4, IL-6, IL-10, and IL-17A were measured. Messenger RNA (mRNA) levels of TGF-ß1, IL-10 and CD4(+) T-cell subtype-specific transcription factors were determined. The expression of CD4(+)CD25(+)forkhead box P3 (FoxP3)(+) cells in lungs and spleens were analyzed. RESULTS: Mice inoculated with PA-laden agarose beads developed chronic PA lung infection during 7-day study period, while mice inoculated with planktonic PA cleared bacteria in 3 days. Compared with mice recovered from acute PA lung infection, those with chronic infection had significantly increased effector T-cell responses, accompanied by a more severe neutrophilic inflammation. Mice with chronic PA lung infection had significantly lower concentration of TGF-ß1 and higher concentrations of IFN-γ, IL-1ß, IL-6, and IL-17A in BAL fluid. Meanwhile, they had significantly lower mRNA levels of TGF-ß1, IL-10 and FoxP3 in lung tissues, and lower expression of CD4(+)CD25(+)FoxP3(+) cells in lungs and spleens. CONCLUSIONS: These findings indicate that Treg cell activity is partly inhibited in mice with chronic PA lung infection, which contributes to the enhanced effector T-cell responses in airways.

Bleomycin-induced pulmonary fibrosis is attenuated by an antibody against KL-6.

BACKGROUND: Elevated levels of KL-6 are reported in the serum and/or bronchoalveolar lavage fluid (BALF) of patients with interstitial lung disease (ILD) and are useful to estimate the severity and prognosis of the disease. However, whether the anti-KL-6 antibody could attenuate pulmonary fibrosis remains unclear. OBJECTIVES: This study aims to investigate the therapeutic effects and mechanisms of anti-KL-6 antibody on bleomycin-induced pulmonary fibrosis. METHODS: A mouse model of pulmonary fibrosis was established by intratracheal injection of bleomycin (5 mg/kg). Mouse received anti-KL-6 antibody (20 ug/day, once a day) from day 7 to 21 after bleomycin injection. The effects of anti-KL-6 antibody were evaluated by pathological examination, measuring hydroxyproline measurements in lung tissues, leukocyte counts in BALF and the expression of collagen type I and type III using qRT-PCR. The expression of profibrotic cytokine (transforming growth factor-ß1, TGF-ß1), antifibrotic cytokine (hepatocyte growth factor, HGF), and KL-6 in lung tissues were analyzed by ELISA. The apoptosis of epithelial cell was examined by TUNEL staining. RESULTS: Anti-KL-6 antibody significantly reduced the number of alveolar inflammatory leukocytes (total and differential counts) in BALF of mice with bleomycin-induced pulmonary fibrosis as well as the content of hydroxyproline in the lung tissues. Treatment with anti-KL-6 antibody downregulated the expression of collagen type I, TGF-ß1 and KL-6, upregulated the expression of HGF and inhibited the apoptosis of epithelial cells. CONCLUSIONS: These findings indicated the anti-KL-6 antibody may potentially be developed as a useful inhibitor of pulmonary fibrosis.

Low-dose clarithromycin therapy modulates CD4(+) T-cell responses in a mouse model of chronic Pseudomonas aeruginosa lung infection.

BACKGROUND AND OBJECTIVE: Low-dose clarithromycin (CAM) is widely used for the treatment of chronic respiratory infections. However, its anti-inflammatory mechanisms have not been fully explored. As CD4(+) T cells play an important role in the initiation of immune responses to infectious microorganisms, we aimed to investigate the effects of low-dose CAM on CD4(+) T-cell responses. METHODS: Fifty-four BALB/c mice were randomly divided into three groups: a control group (inoculated with sterile agarose beads and treated with saline from day 7), a saline group (inoculated with Pseudomonas aeruginosa-loaded beads and treated with saline from day 7) and a CAM group (identical to the saline group, except that saline was replaced by CAM solution). Bronchoalveolar lavage (BAL) fluid cell counts, bacterial load, lung tissue histology and pulmonary CD3(+) CD4(+) cell numbers were assessed. Levels of T helper (Th)1/Th2/Th17 cytokines and suppressor cytokines (interleukin (IL)-10 and transforming growth factor-ß1) were analysed. Messenger RNA (mRNA) levels for transcription factors for CD4(+) T-cell subsets were determined. RESULTS: The CAM group had lower BAL fluid cell counts, pathological scores and pulmonary CD3(+) CD4(+) cell numbers compared with the saline group, whereas the bacterial load was not significantly different. Levels of Th1/Th17 cytokines and expression of a transcription factor for naturally occurring regulatory T cells (Treg) were significantly decreased in the CAM group compared with the saline group, whereas there was no significant difference in GATA-3 mRNA expression. CONCLUSIONS: This study demonstrated a downregulation of Th1/Th17/naturally occurring Treg responses after treatment with low-dose CAM in mice with chronic P. aeruginosa lung infection.

Validity of asthma control test for asthma control assessment in Chinese primary care settings.

OBJECTIVE: To evaluate the validity of the Asthma Control Test (ACT) for assessing clinical asthma control in Chinese patients in primary care settings. METHODS: This multicenter study involved 403 asthma patients from 15 primary care settings in China, who had completed the ACT, Asthma Control Questionnaire (ACQ), and spirometry testing. According to the rating of asthma control by asthma specialists in line with the Global Initiative for Asthma 2006 guidelines, patients were divided into uncontrolled, partly controlled, and controlled groups to evaluate the reliability, empirical validity, and screening accuracy of the ACT. The screening accuracy of the ACT and ACQ was analyzed comparatively, and the asthma control levels rated by the patients and the specialists were also compared. RESULTS: The five-item ACT had an internal consistency reliability of 0.861 and a correlation coefficient with the specialists' rating of 0.697. The ACT scores showed significant differences between different levels of FEV(1) percent predicted (F = 37.59; p < 0.0001) and specialists' ratings of asthma control (F = 169.53; p < 0.0001), and also between patients requiring different treatment adjustments (F = 111.33; p < 0.0001). The asthma was controlled for an ACT score of >/= 20, partly controlled for scores of 19 and 18, and uncontrolled for a score of

Validity of Asthma Control Test in Chinese patients.

BACKGROUND: So far, in China, there has been no effective or easy procedure to define the control of asthma. This study assesses the validity of Asthma Control Test in Chinese patients. METHODS: Three questionnaires (Asthma Control Test, Asthma Control Questionnaire and the 30 second asthma test) were administered to 305 asthma patients from 10 teaching hospitals across China. Spirometry was also used. Asthma specialists rated the control of asthma according to patients' symptoms, medications and forced expiratory volume in first second. The patients were divided into noncontrolled group and controlled group according to the specialists' rating. Reliability, empirical validity and screening accuracy were conducted for Asthma Control Test scores. Screening accuracy was compared among 3 questionnaires. The patients' self rating and the specialists' rating were also compared. RESULTS: The internal consistency reliability of the 5-item Asthma Control Test was 0.854. The correlation coefficient between Asthma Control Test and the specialists' rating was 0.729, which was higher than other instruments. Asthma Control Test scores discriminated between groups of patients differing in the percent predicted forced expiratory volume in first second (F = 26.06, P < 0.0001), the specialists' rating of asthma control (F = 88.24, P < 0.0001) and the Asthma Control Questionnaire scores (F = 250.57, P < 0.0001). Asthma Control Test showed no significant difference with Asthma Control Questionnaire in the percent correctly classified, while the percent correctly classified by Asthma Control Test was much higher than 30 second asthma test. The patients' self rating was the same as assessment of the specialists (t = 0.65, P = 0.516). CONCLUSION: The Asthma Control Test is an effective and practicable method for assessing asthma control in China.