Gut-derived fungemia due to Kodamaea ohmeri combined with invasive pulmonary aspergillosis: a case report.

BACKGROUND: Kodamaea ohmeri is a rare pathogen with high mortality and is found among blood samples in a considerable proportion; however, gastrointestinal infection of K. ohmeri is extremely rare. Invasive pulmonary aspergillosis is also an uncommon fungal; these two fungal infections reported concomitantly are unprecedented. CASE PRESENTATION: We described a case of a 37-year-old male who got infected with K. ohmeri and invasive pulmonary aspergillosis. We used the mass spectrometry and histopathology to identify these two fungal infections separately. For the treatment of K. ohmeri, we chose caspofungin. As for invasive pulmonary aspergillosis, we used voriconazole, amphotericin B, and then surgery. The patient was treated successfully through the collaboration of multiple disciplines. CONCLUSIONS: We speculate that the destruction of the intestinal mucosa barrier can make the intestine one of the ways for certain fungi to infect the human body.

Efficacy and tolerability of budesonide/formoterol added to tiotropium compared with tiotropium alone in patients with severe or very severe COPD: A randomized, multicentre study in East Asia.

BACKGROUND AND OBJECTIVE: Triple combination therapy with tiotropium plus budesonide/formoterol has improved lung function and reduced exacerbation risk in patients with chronic obstructive pulmonary disease (COPD) in Western countries, but no such data exist for East Asian patients. This study aimed to evaluate the efficacy and tolerability of adding budesonide/formoterol to tiotropium compared with tiotropium alone in East Asian patients with severe/very severe COPD. METHODS: This 12-week, randomized, parallel-group, multicentre, open-label study was conducted in East Asia. After a 14-day run-in period during which patients received tiotropium 18 µg once daily, patients were randomized to tiotropium (18 µg once daily) + budesonide/formoterol (160/4.5 µg 2 inhalations twice daily) or tiotropium alone (18 µg once daily). The primary endpoint was change from baseline in pre-dose forced expiratory volume in 1 s (FEV1 ) to the mean of values measured at Weeks 1, 6 and 12. RESULTS: Pre-dose FEV1 significantly increased from baseline with tiotropium plus budesonide/formoterol (n = 287) versus tiotropium alone (n = 291) (5.0% vs 0.6%; treatment difference: 4.4% (95% CI: 1.9-6.9), P = 0.0004). Triple therapy also reduced the COPD exacerbation rate by 40.7% (P = 0.0032) and prolonged time to first exacerbation (38.6% risk reduction, P = 0.0167) versus tiotropium alone and markedly improved health-related quality of life (HRQoL), measured using the St George's Respiratory Questionnaire. Incidence of adverse events was 26% for both groups. CONCLUSIONS: In East Asian patients with severe/very severe COPD, adding budesonide/formoterol to tiotropium was associated with significant improvements in FEV1 and HRQoL and lower COPD exacerbation rates. Treatment was generally well tolerated. CLINICAL TRIAL REGISTRATION: NCT01397890 at Clinicaltrials.gov.

Meta-analysis of the adverse effects of long-term azithromycin use in patients with chronic lung diseases.

The adverse effects of azithromycin on the treatment of patients with chronic lung diseases (CLD) were evaluated in the present study. MEDLINE and other databases were searched for relevant articles published until August 2013. Randomized controlled trials that enrolled patients with chronic lung diseases who received long-term azithromycin treatment were selected, and data on microbiological studies and azithromycin-related adverse events were abstracted from articles and analyzed. Six studies were included in the meta-analysis. The risk of bacterial resistance in patients receiving long-term azithromycin treatment was increased 2.7-fold (risk ratio [RR], 2.69 [95% confidence interval {95% CI}, 1.249, 5.211]) compared with the risk in patients receiving placebo treatment. On the other hand, the risk of bacterial colonization decreased in patients receiving azithromycin treatment (RR, 0.551 [95% CI, 0.460, 0.658]). Patients receiving long-term azithromycin therapy were at risk of increased impairment of hearing (RR, 1.168 [95% CI, 1.030, 1.325]). This analysis provides evidence supporting the idea that bacterial resistance can develop with long-term azithromycin treatment. Besides the increasingly recognized anti-inflammatory role of azithromycin used in treating chronic lung diseases, we should be aware of the potential for adverse events with its long-term use.

NF-κB inhibition after cecal ligation and puncture reduces sepsis-associated lung injury without altering bacterial host defense.

INTRODUCTION: Since the NF-κB pathway regulates both inflammation and host defense, it is uncertain whether interventions targeting NF-κB would be beneficial in sepsis. Based on the kinetics of the innate immune response, we postulated that selective NF-κB inhibition during a defined time period after the onset of sepsis would reduce acute lung injury without compromising bacterial host defense. METHODS: Mice underwent cecal ligation and puncture (CLP). An NF-κB inhibitor, BMS-345541 (50 µg/g mice), was administered by peroral gavage beginning 2 hours after CLP and repeated at 6 hour intervals for 2 additional doses. RESULTS: Mice treated with BMS-345541 after CLP showed reduced neutrophilic alveolitis and lower levels of KC in bronchoalveolar lavage fluid compared to mice treated with CLP+vehicle. In addition, mice treated with CLP+BMS had minimal histological evidence of lung injury and normal wet-dry ratios, indicating protection from acute lung injury. Treatment with the NF-κB inhibitor did not affect the ability of cultured macrophages to phagocytose bacteria and did not alter bacterial colony counts in blood, lung tissue, or peritoneal fluid at 24 hours after CLP. While BMS-345541 treatment did not alter mortality after CLP, our results showed a trend towards improved survival. CONCLUSION: Transiently blocking NF-κB activity after the onset of CLP-induced sepsis can effectively reduce acute lung injury in mice without compromising bacterial host defense or survival after CLP.

Structure and function of OprD protein in Pseudomonas aeruginosa: from antibiotic resistance to novel therapies.

Pseudomonas aeruginosa (P. aeruginosa) is a common pathogen isolated from patients with nosocomial infections. Due to its intrinsic and acquired antimicrobial resistance, limited classes of antibiotics can be used for the treatment of infection with P. aeruginosa. Of these, the carbapenems are very important; however, the occurrence of carbapenem-resistant strains is gradually increasing over time. Deficiency of the outer membrane protein OprD confers P. aeruginosa a basal level of resistance to carbapenems, especially to imipenem. Functional studies have revealed that loops 2 and 3 in the OprD protein contain the entrance and/or binding sites for imipenem. Therefore, any mutation in loop 2 and/or loop 3 that causes conformational changes could result in carbapenem resistance. OprD is also a common channel for some amino acids and peptides, and competition with carbapenems through the channel may also occur. Furthermore, OprD is a highly regulated protein at transcriptional and post-transcriptional levels by some metals, small bioactive molecules, amino acids, and efflux pump regulators. Because of its hypermutability and highly regulated properties, OprD is thought to be the most prevalent mechanism for carbapenem resistance in P. aeruginosa. Developing new strategies to combat infection with carbapenem-resistant P. aeruginosa lacking OprD is an ongoing challenge.

PTPRH Alleviates Airway Obstruction and Th2 Inflammation in Asthma as a Protective Factor.

PURPOSE: PTPRH inhibits EGFR activity directly in cancer patients and activated EGFR induces goblet cell hyperplasia and mucus hypersecretion in asthma. However, the function of PTPRH in asthma remains unknown. The purpose of this study was to access the association of PTPRH with asthma and its underlying mechanism. PATIENTS AND METHODS: We examined the PTPRH level in asthma patients (n = 108) and healthy controls (n = 35), and analyzed the correlations between PTPRH and asthma-related indicators. Human bronchial epithelial cell (HBECs) transfected with PTPRH and asthma mouse model were set up to investigate the function of PTPRH. RESULTS: The expression of PTPRH was significantly increased and correlated with pulmonary function parameters, including airway obstruction, and T-helper2 (Th2) associated markers in asthma patients. PTPRH increased in the house dust mite (HDM)-induced asthmatic mice, while Th2 airway inflammation and Muc5ac suppressed when treated with PTPRH. Accordingly, PTPRH expression was markedly increased in IL-13-stimulated HBECs but PTPRH over-expression suppressed MUC5AC. Moreover, HBECs transfected with over-expressed PTPRH inhibited the phosphorylation of EGFR, ERK1/2 and AKT, while induced against PTPRH in HBECs dephosphorylated of EGFR, ERK1/2 and AKT. CONCLUSION: PTPRH reduces MUC5AC secretion to alleviate airway obstruction in asthma via potential phosphorylating of EGFR/ERK1/2/AKT signaling pathway, which may provide possible therapeutic implications for asthma.

Meta-analysis and systematic review of procalcitonin-guided therapy in respiratory tract infections.

Circulating procalcitonin (PCT) is a biomarker that can be used in diagnosing bacterial infections. We performed a quantitative meta-analysis of available randomized controlled trials to determine whether antibiotic therapy based on PCT measurements alters clinical outcomes and antibiotic use in patients with lower respiratory tract infections. We identified studies through MEDLINE (1996 to 2010), the ISI Web of Knowledge (1996 to 2010), and Ovid. Studies that met our criteria were prospective, randomized controlled trials involving patients with respiratory tract infections. Outcomes of mortality, intensive care unit (ICU) admission, length of hospital stay, number of antibiotic prescriptions, and duration of antibiotic treatment were evaluated. Eight studies randomizing 3,431 patients met our criteria for inclusion. Pooled analysis showed a significant reduction in number of antibiotic prescriptions and duration of antibiotic use in patients with PCT-guided antibiotic treatment compared to standard therapy. In addition, the use of PCT-guided antibiotic therapy did not impact mortality, ICU admission, or length of hospital stay in these studies. A high degree of heterogeneity was identified in 3 of 5 outcomes that were evaluated, and sensitivity analysis indicated that heterogeneity was decreased among studies using the same PCT-based treatment algorithm. In conclusion, PCT-guided antibiotic therapy in patients with respiratory tract infections appears to reduce antibiotic use without affecting overall mortality or length of stay in the hospital.

A CT-derived deep neural network predicts for programmed death ligand-1 expression status in advanced lung adenocarcinomas.

BACKGROUND: Programmed death ligand-1 (PD-L1) expression remains a crucial predictor in selecting patients for immunotherapy. The current study aimed to non-invasively predict PD-L1 expression based on chest computed tomography (CT) images in advanced lung adenocarcinomas (LUAD), thus help select optimal patients who can potentially benefit from immunotherapy. METHODS: A total of 127 patients with stage III and IV LUAD were enrolled into this study. Pretreatment enhanced thin-section CT images were available for all patients and were analyzed in terms of both morphologic characteristics by radiologists and deep learning (DL), so to further determine the association between CT features and PD-L1 expression status. Univariate analysis and multivariate logical regression analysis were applied to evaluate significant variables. For DL, the 3D DenseNet model was built and validated. The study cohort were grouped by PD-L1 Tumor Proportion Scores (TPS) cutoff value of 1% (positive/negative expression) and 50% respectively. RESULTS: Among 127 LUAD patients, 46 (36.2%) patients were PD-L1-positive and 38 (29.9%) patients expressed PD-L1-TPS ≥50%. For morphologic characteristics, univariate and multivariate analysis revealed that only lung metastasis was significantly associated with PD-L1 expression status despite of different PD-L1 TPS cutoff values, and its Area under the receiver operating characteristic curve (AUC) for predicting PD-L1 expression were less than 0.700. On the other hand, the predictive value of DL-3D DenseNet model was higher than that of the morphologic characteristics, with AUC more than 0.750. CONCLUSIONS: The traditional morphologic CT characteristics analyzed by radiologists show limited prediction efficacy for PD-L1 expression. By contrast, CT-derived deep neural network improves the prediction efficacy, it may serve as an important alternative marker for clinical PD-L1 detection.

Targeting the CD134-CD134L interaction using anti-CD134 and/or rhCD134 fusion protein as a possible strategy to prevent lupus nephritis.

Lupus nephritis (LN) is characterized by an increased upregulation of Th1. This study was undertaken to evaluate the role of CD134 in cytokine production in peripheral blood mononuclear cells (PBMCs) from subjects with LN. Percentages of IFN-gamma- (Th1), IL-4-, and IL-10- (Th2) producing cells within the PBMC CD4+ T cell population of LN subjects were found to be higher than those of healthy subjects. Stimulation of PBMC from LN subjects with anti-CD3 epsilon mAb/rIL-2 resulted in further increases in cytokine production. Stimulation in the presence of anti-CD134 mAb resulted in reduced IL-4 and IL-10 production; however, it also resulted in increased IFN-gamma production. Stimulation in the presence of the fusion protein rhCD134:Fc resulted in decreased production of all three cytokines. The possibilities that anti-CD134 therapy may control the extent of IL-4- and IL-10-mediated damage in active LN and that rhCD134:Fc therapy may prevent occurrence of LN are discussed.

[Impact of chronic obstructive pulmonary disease on quality of life and economic burden in Chinese urban areas].

OBJECTIVE: To assess the impact of chronic obstructive pulmonary disease (COPD) on the quality of life and economic burden in Chinese urban areas. METHODS: COPD patients (n = 723) were interviewed face-to-face in outpatient departments in 6 large cities in China. The questionnaire included social and demographic information, current health status, quality of life (SGRQ), and medical expenditure on outpatient visit, hospitalization, medicine purchasing in medicine stores in the last 12 months, and other expenditures related with COPD were also collected. All the data were analyzed using descriptive method. RESULTS: Of the 723 COPD patients interviewed, 73% were male and the average age was 67 years old. The average symptom score of SGRQ was 49 +/- 24, activity score 57 +/- 23, impact score 46 +/- 23 and total score 50 +/- 21, which were all higher than scores of the healthy populations. The average direct medical cost (including outpatient cost, inpatient cost, and medicine purchasing cost) was 11 744 RMB yuan annually. The direct non-medical cost (including transportation fee, nutrition fee, and nursing fee) was 1570 RMB yuan. 36% of the patients in work had an average of 17 working days lost in the last 12 months because of COPD, while 17% of their relatives had an average of 14 working days lost for caring the patients. CONCLUSIONS: COPD has a serious impact on the quality of life of Chinese urban patients and places a heavy economic burden on their family and the society. Management of COPD should be improved for patients at stable conditions, so as to reduce the incidence and exacerbation of COPD.

Tuberculosis in southern Chinese renal-transplant recipients.

OBJECTIVES: To analyze the characteristics of tuberculosis (TB) in Southern Chinese renal transplant recipients, and summarize the corresponding experiences in diagnosis and management. METHOD: Retrospectively study 41 documented post-transplant TB cases out of the 2333 patients who received kidney transplantation in the First Affiliated Hospital of Sun Yat-sen University between Jan. 1991 and Apr. 2007. RESULTS: TB in the post-renal-transplant population in Southern China displayed the following characteristics: (i) high incidence within a short time after transplantation, the median interval between renal transplantation and diagnosis of TB was 8 months (range: 1-156 months) and 56.1% were diagnosed within the first year post-transplant; (ii) high prevalence (51.2%) of extra-pulmonary tuberculosis; (iii) high co-infection rate (19.5%), pathogens included candida albicans, pseudomonas aeruginosa, staphylococcus aureus, Acinetobacter haemolyticus and cytomegalovirus; (iv) fever (82.9%), cough (56.1%) and sputum (39.0%) are the most common clinical manifestations; (v) purified protein derivative of tuberculin (PPD) skin test had little diagnostic value in this group with a negative result in all 41 cases; (vi) acute rejection (29.3%) and liver function damage (17.1%) were the main adverse effects of anti-tuberculosis chemotherapy; (vii) mortality of patients with post-transplant tuberculosis reached up to 22.0%. CONCLUSIONS: Chinese renal transplant recipients face a high risk of TB because of their immuno-compromised state and epidemiological prevalence of the disease. Therefore, attention should be given to this differential diagnosis in clinical practice. Balancing the benefits and disadvantages of anti-tuberculosis chemotherapy is of importance for this specific population.

[Effects of glucocorticoids on tuberculous pleural effusion: experiment with rats].

OBJECTIVE: To explore the effects of systemic glucocorticoid treatment on tuberculous pleural effusion. METHODS: Ninety Wistar rats were intrapleurally injected with 0.03 mg of standard human Mycobacterium tuberculosis to establish models of tuberculous pleural effusions and then were randomly divided into 2 equal groups both without anti-tuberculosis treatment: glucocorticoids group (GG) undergoing intramuscular injection of 0.3 mg triamcinolone acetonide in the right thigh 24 h after intrapleural injection, and control group (CG) received nothing as control. 8, 24, 32, and 48 hours, and 3, 5, 7, 10, and 15 days after intramuscular injection 5 rats from each group were killed. The thorax was opened, the amount of pleural effusion (PE) was recorded, and the pleural cavity, histopathology of pleura and lung parenchyma were examined. The white blood cell (WBC) count and differential leukocyte count, and levels of total protein (TP), glucose (GLU), and lactic dehydrogenase (LDH) in the PE were determined. Bioassays were used to detect the PE levels of soluble intercellular adhesion molecule-1 (sICAM-1), transforming growth factor beta1 (TGF-beta1), and interferon gamma (IFN-gamma). RESULTS: The PE volumes of GG 8 - 48 h after the intramuscular injection were significantly lower than those of CG (P < 0.05, P < 0.01), and PE completely disappeared on day 3. The WBC in PE 24 - 48 h after and the percentages of neutrophils 8 - 48 h after the intramuscular injection of GG were all significant lower than those of CG (all P < 0.01). The TP levels 32 and 48 h after the intramuscular injection of GG were both significantly higher than those of CG (both P < 0.01). The LDH level of GG within 24 h after the intramuscular injection was significantly lower than that of CG (P < 0.01). Both the sICAM-1 and TGF-beta1 levels of GG were higher 8 h after the intramuscular injection, but lower 48 h after the intramuscular injection than those of CG (both P < 0.01). The IFN-gamma levels 8 - 48 h after the intramuscular injection of GG were all higher than those of CC (all P < 0.01). The IFN-gamma/TGF-beta1 ratios at different time points of GG were all higher than those of CG, and there were significant differences in those 8 - 48 h after the intramuscular injection between these 2 groups (all P < 0.01). Pathologically, the mean thickness of pleura in GG was significantly less than that in CG. Congestion and edema in subpleural and pulmonary interstitium were less in GG than in CG. CONCLUSION: Early use of glucocorticoids helps reduce the inflammatory response in pleural cavity in tuberculous pleurisy accelerate the absorption of pleural effusion and decrease the thickness of pleura.

[The effects and therapeutic duration of oral corticosteroids in patients with acute exacerbation of chronic obstructive pulmonary diseases].

OBJECTIVE: To observe the clinical efficacy and therapeutic duration of oral corticosteroids in patients with acute exacerbations of chronic obstructive pulmonary diseases (AECOPD). METHODS: A randomized, double-blinded, placebo-controlled clinical study was designed. One hundred and thirty hospitalized patients with AECOPD were randomly divided into three groups: group 1 (n = 44) received 30 mg/d of prednisone for 7 days and then placebo for another 7 days; group 2 (n = 43) received 30 mg/d of prednisone for 10 days, then tapered to 15 mg/d for 4 days; group 3 (n = 43) received placebo for 14 days. The lung functions, arterial blood gas, length of stay, symptom scores, failure rate of treatment, side-effect of corticosteroids and the rate of relapse were evaluated before and after treatments. The results were analyzed by statistical package for social science (SPSS version 11.0). Measurement data were expressed by (-x) +/- s, and t test was used for the comparison of the data before and after treatments. Chi-square test was used for the comparison of count data. Analysis of variance was applied to test for differences between the three groups. Multiple comparison was analyzed by SNK test. Fisher exact test was used for the comparison of the failure rate of treatment, the rate of relapse and safety evaluation. RESULTS: Compared with the placebo group [group 3, (0.74 +/- 0.32)L], the FEV(1) in [group 1 (0.87 +/- 0.23) L and in group 2 (0.93 +/- 0.30) L] were significantly increased (F = 4.53, P < 0.05). The PaO(2) in group 1 (79 +/- 9) mm Hg (1 mm Hg = 0.133 kPa) and in group 2 (80 +/- 10) mm Hg were also increased significantly [group 3 was (73 +/- 12) mm Hg] (F = 3.98, P < 0.05). The length of stay (LOS) in group 1 (12.5 +/- 3.5) d and in group 2 (12.4 +/- 4.1) d were shortened [group 3 was (13.5 +/- 3.6) d] (F = 3.82, P < 0.05). However, no difference of FEV(1), PaO(2) and LOS was found between 7-day and 14-day durations of corticosteroids therapy. There were no differences in symptom scores, failure rate of treatment, side-effect of corticosteroids and the rate of relapse among the 3 groups. CONCLUSIONS: Oral prednisone results in improvement of FEV(1), PaO(2) and LOS in hospitalized patients with AECOPD. A 7-day or 14-day duration of oral prednisone is of the same clinical efficacy. From these results, we recommend that 30 mg/d of prednisone for a 7-day duration is appropriate for the treatment of AECOPD.

Pseudomonas aeruginosa induces cellular senescence in lung tissue at the early stage of two-hit septic mice.

We presume that severe secondary Pseudomonas aeruginosa (PA) infection can lead to cellular senescence in lung tissue and thus contribute to high mortality. We established a two-hit mouse model using cecal ligation and puncture (CLP) followed by sublethal PA lung infection. In lung tissue, increased infiltration of inflammatory cells, elevated lung injury and augmented cellular senescence was shown in mice with CLP followed by sublethal PA infection, and these observations reached a higher rank when higher (H) loads PA (PAO1) were administered to CLP mice (CLP + PAO1-H). Accordingly, oxidative stress-related element gp91phox and inflammation regulator NF-κB were greatly activated in CLP + PAO1-H mice compared to others. There was no obvious inflammation or cellular senescence in sham control, PAO1-infected mice. Consequently, CLP + PAO1-H mice had the highest expression levels of inflammatory cytokines IL-6, TNFα and iNOS among those groups. There was lower bacterial clearance ability in CLP + PAO1-H mice than in other mice. CLP + PAO1-H only had approximately 10% survival after 7 days of investigation and was much lower than others. In conclusion, higher mortality due to increased lung inflammation and cellular senescence are observed in mice with increased loads of PA infection secondary to CLP.

Using ROS as a Second Messenger, NADPH Oxidase 2 Mediates Macrophage Senescence via Interaction with NF-κB during Pseudomonas aeruginosa Infection.

Pseudomonas aeruginosa (PA) is one of the most prevalent pathogens that cause nosocomial infection in critical patients. However, the mechanisms underlying macrophage growth status and functional changes during PA infection are yet unknown. In the present study, NADPH oxidase, gp91phox (NOX2) mediated macrophage to senescence in a PAO1 colony-dependent manner. gp91phox might regulate the senescence process through mutual interaction with the NF-κB pathway. During infection, the overexpression or downregulation of gp91phox in macrophage could affect the nuclear activity of NF-κB p65, while the downregulation of NF-κB p65 led to a suppressed expression of gp91phox. Reactive oxygen species (ROS) served as the second messenger between both molecules as the ROS inhibitor, N-acetylcysteine (NAC), could partially restore these changes. Consequently, the level of ROS and inflammatory cytokines, including IL-6 and TNFα, elevated during PAO1 infection, and their production altered as a result of the genetic manipulation of gp91phox and NF-κB p65, as well as NAC treatment. Also, the senescent phenotypes, SA-ß-gal staining and p16ink4a, changed after genetic manipulation with gp91phox and NF-κB p65 and NAC treatment. The capacity of phagocytosis in macrophages was decreased during senescence. In conclusion, PA directs the macrophage towards senescence, and senescent macrophages exhibit a decreased ability of phagocytosis. This process of senescence was regulated by the interactions between NADPH oxidase gp91phox and NF-κB p65 via ROS as a second messenger.

Expression of aquaporin-1 in rat pleural mesothelial cells and its specific inhibition by RNA interference in vitro.

BACKGROUND: The discovery of water channel aquaporins (AQPs) has greatly expanded the understanding of the regulation of the water permeability of biological membranes. Aquaporin-1 (AQP1) may be involved in fluid transport in numerous pathological conditions. The objective of the present study was to examine whether AQP1 is present in cultured rat pleural mesothelial cells (PMCs) and to investigate the specific inhibitory effect of RNA interference (RNAi) on AQP1 expression in PMCs, which may provide a new method for the further studies on the relation between expression of AQP1 in PMCs and pleural fluid removal in vivo. METHODS: PMCs were isolated and cultured from rat pleura. The expression of AQP1 in PMCs was confirmed by immunocytochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR). Two eukaryotic expression plasmid vectors of short hairpin RNA (shRNA) specific for the AQP1 gene of rat sapien were designed and constructed. The recombinant plasmid vectors were transfected into cultured rat PMCs by cation liposomes. Flow cytometry was used to screen the most effective shRNA at 48 hours after transfection. The expressions of AQP1 mRNA and protein were detected by RT-PCR and Western blotting method at 48 hours after transfection. RESULTS: RT-PCR and immunostaining revealed that AQP1 mRNA and protein were present in cultured rat PMCs. Two effective eukaryotic expression plasmid vectors of shRNA specific for the AQP1 gene were constructed successfully. The levels of the expression of AQP1 were inhibited by 83.45%, 90.93%, respectively, at mRNA level and 41.24%, 67.60%, respectively at protein level by two recombinant plasmids at 48 hours after transfection. The expression of AQP1 in PMCs transfected with plasmid was significantly lower than that of the cells transfected with the control plasmid HK and that of the untransfected cells (P < 0.01). There was no significant difference in AQP1 expression between the control group and the group transfected with AQP1 nonspecific shRNAs (P < 0.05). CONCLUSIONS: The expression of AQP1 was present in rat PMCs. The application of shRNA-AQP1 could markedly inhibit the expression of AQP1 in cultured rat PMCs. The use of RNAi is a promising tool for future research into the mechanisms of pleural fluid in vivo.

[Effect of hypertonic medium on expression of aquaporin-1 in pleural mesothelial cells: experiment with rats].

OBJECTIVE: To study the expression of aquaporin-1 (AQP-1) in pleural mesothelial cells (PMCs) and the influence of hypertonic medium thereupon. METHODS: Rat PMCs were isolated, cultured, and divided into 2 groups: hypertonic group, cultured in hypertonic media with NaCl of different concentrations for 24 h and control group, cultured in D-MEM/F-12 medium. The 100 mmol/L NaCI group was examined at the time points 6, 12, 18, and 24 h respectively. RT-PCR and Western blotting were used to analyze the mRNA and protein expression of AQP-1. RESULTS: The A values of AQP-1 protein expression were 24.0 +/- 1.8, 27.8 +/- 2.4, 31.7 +/- 2.5, 89.7 +/- 6.2, and 107.7 +/- 9.3 respectively in the PMCs treated with hypertonic media with NaCl of the concentrations of 25, 50, 75, 100, and 150 mmol/L, all significantly higher than that in the control group (10.8 +/- 1.5, all P < 0.01). The A values of AQP-1 protein expression were 42.1 +/- 2.6, 78.9 +/- 3.6, 109.6 +/- 7.6, and 123.4 +/- 8.7 in the PMCs treated with hypertonic media with NaCl of the concentration of 100 mmol/L after 6 h, 12 h, 18 h, and 24 h, all significantly higher than that in the control group (12.9 +/- 1.9, P < 0. 01). The a values of AQP-1 mRNA expression were 62.6 +/- 6.4, 75.3 +/- 5.5, 122.3 +/- 11.4, 196.2 +/- 18.1, and 223.0 +/- 19.3 respectively in the PMCs treated with hypertonic media with NaCl of the concentrations of 25, 50, 75, 100, and 150 mmol/L, all significantly higher than that in the control group (23.5 +/- 2.7, all P < 0.01). The A values of AQP-1 protein expression were 83.6 +/- 6.1, 159.3 +/- 6.9, 266.2 +/- 24.1, and 196.2 +/- 15.7 in the PMCs treated with hypertonic media with NaCl of the concentration of 100 mmol/L after 6 h, 12 h, 18 h, and 24 h, all significantly higher than that in the control group (26.5 +/- 2.8, P < 0.01). CONCLUSION: Hypertonic medium increases the expression of AQP-1 mRNA and protein. AQP-1 participates in the pleural fluid formation.

[Inhibition of the aquaporin-1 gene expression by RNA interference: experiment with cultured rat pleural mesothelial cells].

OBJECTIVE: To investigate the influence of RNA interference (RNAi) on the expression of aquaporin-1 (AQP1) gene and to investigate the feasibility of gene therapy for pleural effusion. METHODS: Two recombinant plasmids with shRNAs targeting the AQP1 gene, AQP1-1-pGenesil and AQP1-2-pGenesil-1 were constructed. Pleural mesothelial cells were obtained from rats, cultured, and randomly divided into 5 groups: blank control group, Lipofectamine 2000 control group, HK negative control group, AQP1-1-pGenesil-1 transfected group, and AQP1-2-pGenesil-1 transfected group. RT-PCR and Western blotting were used to detect the mRNA and protein expression of AQP1. RESULTS: The mRNA expression levels of aquaporin-1 of the AQP1-1-pGenesil-1 and AQP1-2-pGenesil-1 transfected groups were inhibited by 83.5% and 90.9% respectively, and the protein expression levels of the AQP1-1-pGenesil-1 and AQP1-2-pGenesil-1 transfected groups were inhibited by 41.2% and 67.6% respectively. CONCLUSION: RNAi can successfully inhibit the expression of AQP1 and has the feature of sequence correlation of shRNA in the cultured rat pleural mesothelial cells. It may be used as a potential new approach for gene therapy of pleural effusion.

Macrolide-resistant Mycoplasma pneumoniae prevalence and clinical aspects in adult patients with community-acquired pneumonia in China: a prospective multicenter surveillance study.

BACKGROUND: Drug resistant Mycoplasma pneumoniae (MP) is a rising issue in the management of community-acquired pneumonia (CAP). Epidemiological monitoring is essential for identifying resistant patterns of MP isolates against various antibiotics in adult CAP patients. METHODS: This is a prospectively designed multicenter study conducted on adult patients with CAP visiting six teaching hospitals in the cities of Beijing, Shanghai and Guangzhou between September 2010 and June 2012. RESULTS: A total of 520 adult patients (mean age: 45.7±26.2 years) with CAP visiting teaching hospitals in the cities of Beijing, Shanghai and Guangzhou were included. Of the 520 patients, only 75 (14.42%) were confirmed MP positive by means of culture and real-time PCR methods. Quinolones were the most common initially prescribed antimicrobial, followed by ß-lactams and ß-lactams plus quinolones. Macrolide resistance was as high as 80% and 72% against erythromycin (ERY) and azithromycin (AZM) respectively, which were associated with the A2063G transition mutation in domain V of the 23S ribosomal RNA (rRNA) gene. Six strains with mild to moderate ERY-resistant level were still susceptible to AZM. Tetracycline (TET), minocycline (MIN) and quinolones [moxifloxacin (MOX) and fluoroquinolones] had no signs of resistance. CONCLUSIONS: High resistance was observed with macrolides, whereas, none of the MP strains were resistant to fluoroquinolones and TET. Hence, macrolide resistant MP (MRMP)_infections could be well treated with fluoroquinolones. However, few isolated strains had minimal inhibitory concentration (MIC) values on the edge of resistance to quinolones, alarming a quinolone-resistant MP in the near future.

[Effect of anti-vascular endothelial growth factor antibody on pleurodesis induced by transforming growth factor-beta or doxycycline in rabbits].

OBJECTIVE: The intrapleural injection of transforming growth factor-beta (TGF-beta) or doxycycline produces excellent pleurodesis in rabbit models. However, the intrapleural injection of these agents induces large pleural effusion which is possibly related to vascular endothelial growth factor (VEGF). This study investigated whether anti-VEGF antibody has any effect on the fluid production or the pleurodesis induced by TGF-beta or doxycycline in rabbits. METHODS: Two groups of New Zealand white rabbits (7 each) were given TGF-beta 5.0 microg intrapleurally. The TGF-beta treatment group received anti-VEGF antibody 10 mg/kg intravenously 24 h before TGF-beta injection and the TGF-beta control group received no antibody. Another two groups of New Zealand white rabbits (7 each) were given doxycycline 10 mg/kg intrapleurally after chest tube placement. The doxycycline treatment group received 10 mg/kg anti-VEGF antibody intravenously 24 h before doxycycline injection and the doxycycline control group received no Anti-VEGF antibody. The rabbits were sacrificed at 2 weeks and the pleurodesis score was graded macroscopically on a 1-8 scale. The degree of angiogenesis in pleural tissues was assessed by immunohistochemical staining for factor VIII which was assessed by computer-assisted digital analysis. RESULTS: The administration of anti-VEGF antibodies had no effect on pleural fluid volume or the characteristics of the fluid. The mean pleurodesis score of TGF-beta control group (7.7 +/- 0.8) was significantly (P < 0.05) higher than that of the antibody pre-treatment TGF-beta group (4.4 +/- 2.4). The mean pleurodesis score of the doxycycline control group (6.0 +/- 1.7) was significantly (P < 0.05) higher than that of the antibody pre-treatment doxycycline group (2.0 +/- 0.9). The administration of the anti-VEGF antibody also reduced the angiogenesis. The percentage of pleural tissue demonstrating angiogenesis in the TGF-beta control group (4.9 +/- 0.4)% was significantly (P < 0.05) higher than that of the antibody treatment TGF-beta group (2.9 +/- 0.7)%. The percentage of pleural angiogenesis in the doxycycline control group (6.9 +/- 2.2)% was significantly (P < 0.05) higher than the antibody pre-treatment doxycycline group (2.2 +/- 0.9)%. CONCLUSIONS: Anti-VEGF antibody significantly inhibits the pleurodesis induced by doxycycline or TGF-beta. This observation suggests that VEGF and angiogenesis play a pivotal role in the production of pleurodesis.