The study of the ratio and distribution of Th17 cells and Tc17 cells in asthmatic patients and the mouse model.

BACKGROUND: Whether CD8+ IL-17-producing T cells, namely Tc17 cells, play a role in asthma has not been determined. The aim of this study was to evaluate and compare the frequency of peripheral blood Th17 cells and Tc17 cells in asthmatic patients. In addition, the number, ratio and distribution of Th17 cells and Tc17 cells in the lung tissue and splenocytes of asthmatic mice were also investigated. METHODS: Th17 and Tc17 cells in the peripheral blood samples of asthmatic patients and in murine spleens were detected by flow cytometric analysis. Th17 and Tc17 cells in murine lung tissues were detected by double immuno-fluorescence stain. IL-17A levels in murine bronchoalveolar lavage were detected by ELISA. RESULTS: The result of the flow cytometric analysis showed the percentage of Th17 cells among CD3+ T cell populations in patients with asthma was higher than that in healthy controls (P < 0.01), The percentage of Tc17 cells was also higher (P < 0.05). The percentages of Th17 and Tc17 cells in asthmatic mice were both much higher than that in control animals (P < 0.01). Frozen sections of lung tissue showed that the number of Th17 cells and Tc17 cells in the asthma group were all significantly higher than in the control group (P < 0.01). CONCLUSIONS: Our findings suggest a functional disequilibrium of Th17 and Tc17 cell subsets in asthma that may contribute to the inflammatory process and provide novel insights into a hypothetical driving role of those cells in disease pathogenesis.

[Adult hospital acquired pneumonia: a multicenter study on microbiology and clinical characteristics of patients from 9 Chinese cities].

OBJECTIVE: To investigate the etiology and clinical characteristics of hospital-acquired pneumonia (HAP) in China and to provide evidence for appropriate therapy. METHODS: We performed a prospective multicenter study in 13 Chinese urban tertiary hospitals. All HAP cases diagnosed at respiratory general ward and respiratory intensive care unit (RICU) from August 2008 to December 2010 were studied. Epidemiological data, etiology and clinical characteristics of enrolled patients were collected. Sputum or tracheal aspirate and blood cultures, Legionella antibodies and Streptococcus pneumoniae urinary antigen tests were performed. Bacteria to antimicrobial susceptibility test was performed. RESULTS: A total of 610 cases of HAP were diagnosed during the study, with an overall incidence of 1.4% among 42 877 hospitalized patients, while the incidence was 0.9% (362/41 261) in respiratory general ward and 15.4% (248/1616) in RICU. 93.9% (573 cases) of patients had at least one underlying disease, and 91.0% (555 cases) had exposure to at least one antimicrobial agent within 90 days prior to HAP diagnosis. Pathogens were identified in 487 patients, with Acinetobacter baumannii [30.0% (183/610)], Pseudomonas aeruginosa [22.0% (134/610)], Staphylococcus aureus [13.4% (82/610)] and Klebsiella pneumonia [9.7% (59/610)] being the most common pathogens. Eighteen patients (3.0%) had infection with fastidious bacteria. A. baumannii and S. aureus were the more frequent pathogens in the ventilator-associated pneumonia (VAP) cases [50.5% (97/192) and 21.4% (41/192)] as compared to non-VAP cases [20.6% (86/418) and 9.8% (41/418), P < 0.01]. A. baumannii and S. aureus were also frequent pathogens in cases with a score of more than 20 by the acute physiology and chronic health evaluation II (APACHEII) scoring [45.7% (69/151) and 20.5% (31/151)], as compared to cases with a score of less than 20 of APACHE II [24.8% (114/459) and 11.1% (51/459), P < 0.01]. A. baumannii showed high resistance rates to carbapenems [more than 70% (109/142)], and the susceptibility to cefoperazone/sulbactam, polymyxin B and tigecycline were 40.8% (58/142), 99.3% (141/142) and 95.8% (136/142) respectively. Resistance rates of P. aeruginosa to meropenem and imipenem were 48.8% (40/82) and 70.7% (58/82) respectively. Methicillin-resistant S. aureus (MRSA) accounted for 87.8% (43/49) in all strains of S. aureus. Mortality rate of VAP cases was 34.5% (61/177), significantly more than that of HAP patients [22.3% (135/605), P < 0.05]. The average hospital stay of patients with HAP was (23.8 ± 20.5) days, significantly more than that of the average for inpatients [(13.2 ± 13.6) days, P < 0.01] during the study period. Mean costs of HAP were (108 950 ± 116 608) yuan, significantly higher than the average hospital costs of respiratory inpatients (17 999 ± 33 364) yuan. CONCLUSIONS: Among Chinese patients hospitalized in urban tertiary medical centers, HAP incidence and mortality rate were high, which increased the patients' hospital stay and the medical costs. Common pathogens were A. baumannii, P. aeruginosa, S. aureus and K. pneumonia. The common bacteria of HAP in China showed high resistance rates to antibiotics.

Recombinant adeno-associated virus vector-mediated delivery of antisense interleukin-5 gene attenuates airway remodeling in allergic rats.

BACKGROUND: Increasing evidence indicates that eosinophils contribute greatly to airway remodeling in asthma. Since interleukin-5 (IL-5) plays a critical role in the regulation of eosinophils in asthma, anti-IL-5 therapy may be a novel approach to inhibit airway remodeling in asthma. OBJECTIVES: In this study, we applied a recombinant adeno-associated virus vector-mediated antisense IL-5 gene delivery (rAAV-ASIL-5) system to investigate its effect on airway remodeling in ovalbumin (OVA)-sensitized and -challenged rats. METHODS: rAAV-ASIL-5 was used to infect OVA-sensitized and -challenged rats. IL-5 protein in bronchoalveolar lavage fluid (BALF) was detected by ELISA. The eosinophils in BALF were counted. Transforming growth factor (TGF)-ß1- and TGF-ß2-positive cells in the peribronchial space were detected by immunohistochemical staining. Lung tissue was collected for Sirius red staining and histological analysis. RESULTS: rAAV-ASIL-5 significantly decreased the level of IL-5 protein, the number of eosinophils in BALF and the numbers of TGF-ß1- and TGF-ß2-positive cells in the peribronchial space. The area of Sirius red staining in airways was also decreased. Moreover, the rAAV-ASIL-5 treatment inhibited the increase in total bronchial wall area and airway smooth muscle area. CONCLUSION: These results suggest that rAAV-ASIL-5-based gene therapy could be used to inhibit airway remodeling in allergic rats.

Increased expression of aryl hydrocarbon receptor and interleukin 22 in patients with allergic asthma.

OBJECTIVES: We sought to determine whether the aryl hydrocarbon receptor (AhR) and interleukin (IL)-22 may be involved in the pathogenesis of the peripheral blood mononuclear cells (PBMCs) in allergic asthmatic patients and whether their expression may be related to the severity of the disease. METHODS: Blood samples were obtained from each subject with allergic asthma (n =18), controlled asthma (n = 17) and healthy controls (n = 12) respectively. The PBMCs were collected for AhR mRNA detection by real-time quantitative polymerase chain reaction (PCR). The plasma was collected for IL-22 protein detection by enzyme-linked immunosorbent assay (ELISA). RESULTS: The expression of AhR mRNA in PBMCs and IL-22 protein in plasma of patients with allergic asthma were higher than those in controlled asthma cases and healthy controls. The plasma concentrations of IL-22 had negative correlation with the predicted percentage of forced expiratory volume in the first second (FEV1%) and the percentage of FEV1 and forced vital capacity (FEV1/FVC%) and it was positively correlated with the asthma severity score (ASS) of the asthmatics. CONCLUSION: Our results suggested that both AhR and IL-22 might be involved in the pathogenesis of allergic asthma in human and the level of IL-22 might have some relationship with the severity of the disease.

Comparative study on the efficacy of tiotropium bromide inhalation and oral doxofylline treatment of moderate to severe stable chronic obstructive pulmonary disease.

This study compared the efficacy and safety of tiotropium bromide inhalation powder (spiriva) and doxofylline oral tablet (doxofylline) in the treatment of chronic obstructive pulmonary disease (COPD). A multi-center, randomized, double-blind, double-dummy, parallel-controlled study involved 127 eligible stable moderate to severe COPD patients treated with inhaled tiotropium dry powder (18 µg/day) or oral doxofylline tablets (0.2 g/time, 2 times a day) for 12 and 24 weeks. Before and after treatment for 12 weeks and 24 weeks, respectively, pulmonary function, 6-min walking distance and dyspnea index were recorded. The results showed that in both tiotropium group and doxofylline groups, after 12-week treatment, FEV(1), FEV(1)/FVC% and 6-min walk distance were significantly higher than those before the medication, while dyspnea index decreased as compared with that before treatment. After 24-week treatment, a slight improvement in the measures was observed as compared with that of 12-weeks treatment, but the difference was not statistically significant. With both 12-week and 24-week treatment, the effect of tiotropium was slightly better than that of doxofylline tablets, with the difference being statistically insignificant. The major adverse events in the tiotropium group and doxofylline group were observed in 9 cases (9.9%) and 12 cases (12.9%), respectively, and no statistically significant difference was found between them. We are led to conclude that both tiotropium at 18 µg a day and doxofylline tablets at 0.2 g/day (two times a day) are effective and safe for the treatment of COPD.

[A multicentre retrospective study of pulmonary mycosis clinically proven from 1998 to 2007].

OBJECTIVE: To investigate the pathogens, clinical manifestations, prognosis of and the risk factors for pulmonary mycosis in China. METHODS: All cases of pulmonary mycosis from 16 centers in 10 cities from Jan. 1998 to Dec. 2007 that met the diagnostic criteria were included for clinical, microbiological and radiological analysis. RESULTS: Totally 474 cases of pulmonary mycosis were retrieved. The top 5 pulmonary mycosis was pulmonary aspergillosis (180 cases, 37.9%), pulmonary candidiasis (162 cases, 34.2%), pulmonary cryptococcosis (74 cases, 15.6%), pneumocystis carinii pneumonia (23 cases, 4.8%) and pulmonary mucormycosis (10 cases, 2.1%). The constituent ratio in the last 3 years was similar to that in the former 7 years. The main pathogens of pulmonary candidiasis were Candida albicans (308/474, 65.0%) and Candida tropicalis (57/474, 12.0%), which were sensitive to common azoles. Compared with bacterial pneumonia, pulmonary mycosis showed more symptoms of hemoptysis (147/474, 31.0%) and pleural effusion (95/474, 20.0%), and less radiological specificity. Classical halo sign (4/474, 0.8%) and crescentic sign (17/474, 3.6%) were only shown in several cases of pulmonary mycosis. The most common underlying diseases were tumor (including solid tumor and malignant hematological diseases) (94/474, 19.8%), chronic obstructive pulmonary disease (52/474, 11.0%), pulmonary tuberculosis (50/474, 10.5%) and diabetes (48/474, 10.1%). Compared with the other common pulmonary mycosis, pulmonary cryptococcosis affected younger patients, and more cases were community-acquired, but fewer cases with underlining diseases or compromised immune function, and had a better prognosis. CONCLUSION: The ahead five species of pulmonary mycosis in China were orderly pulmonary aspergillosis, pulmonary candidosis, pulmonary cryptococcosis, pneumocystis carinii pneumonia and pulmonary mucormycosis. The main pathogens of pulmonary candidosis were Candida albicans and Candida tropicalis, which were sensitive to common azoles. Compared with the other common pulmonary mycosis, pulmonary cryptococcosis catch younger patients, had more community-acquired cases, and had better prognosis.

Effectiveness of omalizumab in patients with severe allergic asthma: A retrospective study in China.

BACKGROUND: We conducted the first real-world study of treatment with omalizumab, a humanized monoclonal anti-immunoglobulin E antibody, in Chinese patients with severe allergic asthma. OBJECTIVE: The primary objective was the steroid-sparing effect of omalizumab after 12 and 16 weeks of treatment. Characteristics of the patient population, treatment patterns, response rate, and other measures of therapeutic effectiveness were also reported. METHODS: This nationwide, retrospective, real-world study was conducted in patients with severe allergic asthma who were treated with omalizumab in China. Data, including demographics, Asthma Control Test (ACT) and laboratory and lung function test results, and omalizumab use information, were extracted from patient records collected as part of a previously conducted real-world survey (Asthma Group of the Respiratory Disease Society of the Chinese Medical Association). RESULTS: In total, 139 patient records were included; 131 and 118 patients remained on treatment at the ≥12- and ≥16-week time points, respectively. The mean ± standard deviation age and median asthma duration (interquartile range) were 47.4 ± 14.3 and 7 (4, 15) years, respectively; 75.6% of patients had a history of allergic disease. Reductions (versus baseline) in inhaled corticosteroid/long-acting ß2 agonists or oral corticosteroids were reported in 61.1% and 63.6% of patients at ≥12 and ≥ 16 weeks, respectively. There were significant improvements in ACT scores (6.08, P < .001) and nitric oxide fraction in exhaled air (-13.0, P = .01) from baseline. Multivariate analysis revealed that age and allergic medical history were predictors of omalizumab treatment response. No serious adverse events were reported. CONCLUSION: Real-world omalizumab treatment was efficacious and well-tolerated in Chinese patients with severe allergic asthma.

Administration of nonviral gene vector encoding rat beta-defensin-2 ameliorates chronic Pseudomonas aeruginosa lung infection in rats.

BACKGROUND: Beta-defensin-2 (BD-2) plays an important role in host defense against pathogenic microbe challenge by its direct antimicrobial activity and immunomodulatory functions. The present study aimed to determine whether genetic up-regulation of rat BD-2 (rBD-2) could ameliorate chronic Pseudomonas aeruginosa lung infection in rats. METHODS: Plasmid-encoding rBD-2 was delivered to lungs in vivo using linear polyethylenimine at 48 h before challenging with seaweed alginate beads containing P. aeruginosa. Macroscopic and histopathological changes of the lungs, bacterial loads, inflammatory infiltration, and the levels of cytokines/chemokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, kertinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2)] were measured at 3 and 7 days post-infection (p.i.). RESULTS: The overexpression of rBD-2 resulted in a significant increase in animal survival rate (at 3 days p.i.), a significant decrease in bacterial loads in the lungs (at 3 and 7 days p.i.), and significantly milder lung pathology. In addition, the overexpression of rBD-2 led to increased infiltration of polymorphonuclear neutrophils (PMN), and elevated protein expression of cytokines/chemokines (IL-1beta, TNF-alpha, KC and MIP-2) at the early stage of infection (at 3 days p.i.), at the same time as being dramatically decreased at the later stage of infection (at 7 days p.i.). CONCLUSIONS: Genetic up-regulation of rBD-2 increased animal survival rate, and reduced bacterial loads in lungs after bacterial infection. The overexpression of rBD-2 also modulated the production of several cytokines/chemokines and increased PMN recruitment at the early stage of infection. Our findings indicate that the enhancement of BD-2 may be an efficacious intervention for chronic P. aeruginosa lung infection.

The effects of antisense interleukin-4 gene transferred by recombinant adeno-associated virus vector on the airway remodeling in allergic rats.

BACKGROUND: Th2-derived cytokines, including interleukin-4 (IL-4), are considered to play an important role in the development of airway remodeling of asthma. OBJECTIVES: Our previous study has demonstrated that a recombinant adeno-associated virus containing antisense against IL-4 gene (rAAV-asIL4) vector could significantly suppress the expression of IL-4 protein and airway inflammation in the rat models of allergic asthma. In this study, we applied the rAAV-asIL4 vector to allergic rats to investigate the effects of anti-IL4 therapy on airway remodeling in allergic asthma. METHODS: rAAV-asIL4 was used to infect the ovalbumin (OVA)-sensitized and challenged rats by tail-vein injection. IL-4 protein in bronchoalveolar lavage fluid (BALF) was detected by enzyme-linked immunosorbent assay. The number of eosinophils in BALF was counted. Transforming growth factor-beta1 (TGF-beta1) and TGF-beta2-positive cells in the peribronchial space were detected by immunohistochemical staining, and collagen deposition beneath the basement membrane was detected by Sirius red stain. The lung tissues were collected for histologic analysis of total bronchial wall area (W(At)) and airway smooth muscle area (W(Am)). RESULTS: rAAV-asIL4 significantly decreased IL-4 protein in BALF of OVA-sensitized and challenged rats. The number of eosinophils in BALF, the TGF-beta1 and TGF-beta2-positive cells in the peribronchial space were also suppressed. Moreover, the rAAV-asIL4 treatment inhibited the area of Sirius red staining in airways and the increase in W(At) and W(Am). CONCLUSION: These results suggest that rAAV-asIL4 may attenuate the airway remodeling process relevant to the inhibition of airway inflammation. This study provides elementary evidence for the potential utility of rAAV-asIL4 as an approach to gene therapy for asthmatic airway remodeling.

Primary pulmonary coccidioidomycosis in China.

Coccidioidomycosis is a fungal infection endemic to south-west USA, north Mexico and parts of Central and South America. We report here a case of primary pulmonary coccidioidomycosis in a previously healthy 14-year-old boy in China, which is considered a non-endemic country. The patient had non-specific symptoms of pulmonary infection, including fever, non-productive cough and night sweats. Both spherules and endospores of Coccidioides immitis were seen histologically following transbronchial biopsy of a cavitary lesion. The patient was treated with amphotericin B and fluconazole. Follow up 6 months post discharge found that the patient made a good recovery.

Expression of the Th1-specific cell-surface protein Tim-3 increases in a murine model of atopic asthma.

T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) is preferentially expressed on Th1-helper type T-cells and functions to repress the Th1-mediated immune response. However, the role of Tim-3 during the inflammatory pathogenesis of asthma remains unclear. This study determines the expression level of Tim-3 in CD4+ T-cells within the peripheral blood and bronchoalveolar lavage fluid (BALF) isolated from a murine model of atopic asthma and explores the potential role of Tim-3 during the inflammatory response. Mice were randomly divided into normal control, asthma day 1, and asthma day 7 groups, and peripheral blood T lymphocytes and BALF cells were collected. The ratio of Tim-3+/CD4+ cells among the total CD4+cell populations from peripheral blood and BALF was determined by flow cytometry, and the expression of the Tim-3 mRNA was determined by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). In contrast with the normal control group, the ratio of Tim-3+/CD4+:CD4+ cells and the level of Tim-3 mRNA in both the peripheral blood T lymphocytes and BALF cells among the asthma day 1 and asthma day 7 groups were significantly increased (p < 0.01), and those in the asthma day 7 group were higher than the asthma day 1 group (p < 0.05). There was also a positive correlation between the ratio of Tim-3+/CD4+:CD4+ detected in BALF and that the ratio detected in peripheral blood T lymphocytes (r = 0.84, p < 0.01). Therefore, the expression of Tim-3 is increased in CD4+ T-cells following airway challenge and likely affects asthma-induced inflammation by repressing the Th1-mediated immune response.

Altered surfactant protein A gene expression and protein homeostasis in rats with emphysematous changes.

BACKGROUND: The decrease of surfactant protein (SP) secreted by the alveolar type II cell is one of the important causes of limiting air of pulmonary emphysema. However, the SP-A gene and protein changes in this disease are rarely studied. This study was undertaken to investigate alterations in SP-A gene activity and protein, and to explore their roles in the pathogenesis of emphysematous changes. METHODS: Twenty Wistar rats were divided randomly into a normal control group (n = 10) and a cigarette smoking (CS) + lipopolysaccharide (LPS) group (n = 10). Ultra-structural changes were observed under an electron microscope. The number of cells positive for SP-A was measured by immunohistochemistry. The mRNA expression and protein level of SP-A in the lung tissues were determined by quantitative polymerase chain reaction (qPCR) and Western blot separately. The protein level of SP-A in lavage fluid was determined by Western blot. RESULTS: The number of cells positive for SP-A of the CS + LPS group (0.35 +/- 0.03) was lower than that of the blank control group (0.72 +/- 0.06, P < 0.05). The level of SP-A in the lung tissues of rats in the CS + LPS group (0.2765 +/- 0.0890) was lower than that in the blank control group (0.6875 +/- 0.1578, P < 0.05). The level of SP-A in the lavage fluid of rats in the CS + LPS group (0.8567 +/- 0.1458) was lower than that in the blank control group (1.3541 +/- 0.2475, P < 0.05). The lung tissues of rats in the CS + LPS group showed an approximate increase (0.4-fold) in SP-A mRNA levels relative to beta-actin mRNA (P < 0.05). CONCLUSIONS: The changes of SP-A may be related to emphysematous changes in the lung. And cigarette smoke and LPS alter lung SP-A gene activity and protein homeostasis.

The alteration and significance of surfactant protein A in rats chronically exposed to cigarette smoke.

In order to confirm the alteration and significance of cigarette smoke exposure on SP-A in rats, 20 Wistar rats were assigned randomly to two groups: an N group (n=10), and an S group (n=10). The ultra-structural change was observed by electron microscopy. The number of cells positive for SPA was by immunohistochemically measured. The mRNA expression in the lung tissues was determined by reverse transcription polymerase chain reaction (RT-PCR). The number of cells positive for SPA of the S group (0.52 +/- 0.05) was lower than that of the N group (0.72+/-0.06) (P<0.05). The levels of mRNA of SPA in the lung tissues of the S group (0.3522+/-0.0512) was significantly lower than that of the N group (0.4432+/-0.05628) (P<0.05). It is concluded that cigarette smoke alone decreased the level of SP-A and that might have an important effect on surfactant metabolism and the host defense functions of surfactant in the peripheral airways, which might play a crucial role in the development of chronic obstructive lung disease.

The efficacy and safety of combination salmeterol (50 microg)/fluticasone propionate (500 microg) inhalation twice daily via accuhaler in Chinese patients with COPD.

BACKGROUND: Few studies of the efficacy and safety of therapy with combinations of salmeterol/fluticasone propionate (SFCs) have been conducted in Chinese patients with COPD, and the benefits of combination therapy in nonsmoking patients with COPD are, to our knowledge, not known. STUDY OBJECTIVES: The aims were to establish the efficacy and tolerability of the therapy with SFC (salmeterol, 50 microg/fluticasone, 500 microg, twice daily) in the management of Chinese COPD patients and to investigate the effectiveness of SFC in nonsmokers with COPD. METHODS AND PATIENTS: This was a randomized, double-blind, placebo-controlled, parallel-group, multicenter study. Changes in prebronchodilator and postbronchodilator FEV(1), quality of life determined by the St. George Respiratory Questionnaire (SGRQ) scores, relief bronchodilator use, nighttime awakenings, and frequency of exacerbations of COPD were measured in patients randomized to receive SFC (n = 297) or placebo (n = 148). Never-smokers, former smokers, and current smokers accounted for 11.7%, 66.7%, and 21.6%, respectively, of the study population. RESULTS: After 24 weeks, the mean changes in prebronchodilator and postbronchodilator FEV(1) were 180 mL (95% confidence interval [CI], approximately 91 to 268; p < 0.001) and 65 mL (95% CI, approximately 14 to 115; p = 0.012), respectively, greater for the SFC group than that for the placebo group. The differences in response to treatment were significant (all p < 0.0001) in former or current smokers but not in never-smokers (p > 0.05). The mean improvement in the total SGRQ score for the SFC group was 5.74 (p < 0.01) greater than that for the placebo group. SFC significantly reduced the frequency of nighttime awakenings and the use of relief bronchodilator. The adjusted ratio of exacerbations of COPD for the SFC group relative to the placebo group was 0.61 (95% CI, approximately 0.45 to 0.84; p < 0.01). There were no significant differences between the SFC and placebo groups in safety measures. CONCLUSIONS: SFC therapy achieved sustained improvement in lung function, quality of life, and control of symptoms, and was well tolerated in Chinese patients. Greater improvements in lung function were found only for COPD patients with a history of smoking. TRIAL REGISTRATION: http://ctr.gsk.co.uk/Summary/fluticasone_salmeterol/studylist.asp Identifier: No. SCO100540.

Effect of intramuscular injection of hepatocyte growth factor plasmid DNA with electroporation on bleomycin-induced lung fibrosis in rats.

BACKGROUND: So far, there is no efficient treatment for pulmonary fibrosis. The objective of this study was to determine whether intramuscular injection of the hepatocyte growth factor (HGF) plasmid DNA by in vivo electroporation could prevent bleomycin-induced pulmonary fibrosis in rats, and to investigate the possible mechanisms. METHODS: Twenty male Wistar rats were randomly divided into four groups: control group (group C), model group (group M), early intervention group (group I) and late intervention group (group II). Groups M, I and II were intratracheally infused with bleomycin, then injected the plasmid pcDNA3.1-hHGF to group I on day 7, 14 and 21. Group II received the same treatment like Group I on day 14 and 21. All the rats were killed on day 28 after bleomycin injection. We detected Homo HGF expression in the rats with ELISA method and estimated the pathological fibrosis score of lung tissue using hematoxylin eosin (HE) and Massion staining. The mRNA expression of transforming growth factor-beta1 (TGF-beta1), cycloxygenase-2 (COX-2), and rat HGF in rat pulmonary parenchyma were evaluated by RT-PCR. Immunohistochemistry and Western blotting were performed to determine the protein expression of transforming TGF-beta1 and COX-2 in lung parenchyma. RESULTS: The plasmid pcDNA3.1-hHGF could express hHGF in NIH3T3 cells and the hHGF protein is secreted into the culture medium. The expression of hHGF protein could be monitored in quadriceps muscle, plasma and lung in Groups I and II. Pulmonary fibrosis levels of Groups I and II were obviously lower than that of group M (P < 0.05). Expression of TGF-beta1 protein and mRNA in lung tissue was markedly decreased in Groups I and II compared with Group M (P < 0.05). The level of expression of HGF and COX-2 mRNA was higher in Groups I and II than in Group M (P < 0.05). CONCLUSIONS: Injection of the plasmid pcDNA3.1-hHGF into skeletal muscle with electroporation has a potential role in the treatment of bleomycin-induced lung fibrosis. Exogenous HGF may inhibit the expression of TGF-beta1 and regulate the crosstalk between AECs and mesenchymal fibroblasts.

Primary culture of alveolar epithelial type II cells and its bionomic study.

To establish a better method of primary culture for alveolar epithelial type II cells (AEC II) and to study its bionomics, alveolar epithelial type II cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin G. The purified AEC II were identified by alkaline phosphatase staining, electron microscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC II could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2-3 x 10(7), and a purity of about 75%-84%. Cells could be quickly identified with AKP staining. AEC II were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC II, and AKP staining is simple in cell identification. AEC II can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.

Clinical value of vascular endothelial growth factor combined with interferon-gamma in diagnosing malignant pleural effusion and tuberculous pleural effusion.

In order to investigate the clinical value of vascular endothelial growth factor (VEGF) combined with interferon-gamma (IFN-gamma) in diagnosing malignant pleural effusion and tuberculous pleural effusion, 42 cases of malignant pleural effusion and 45 cases of tuberculous pleural effusion in Tongji Hospital, from March 2004 to May 2005, were included. The carcinoembryonic antigen (CEA), VEGF and IFN-gamma levels of pleural effusion were detected by using ELISA, and adenosine deaminase (ADA) activity was determined by using enzyme kinetic analytical method. The sensitivity, specificity, accuracy and area under the curve (AUC(ROC)) of CEA and VEGF, VEGF/IFN-gamma ratio, ADA and IFN-gamma were measured by receiver operating characteristic curve (ROC). The results showed that CEA, VEGF levels and VEGF/IFN-gamma ratio were significantly higher and the ADA and IFN-gamma levels were significantly lower in malignant group than those in tuberculous group (P<0.01). The sensitivity, specificity, accuracy and AUC(ROC) of VEGF/IFN-gamma ratio (88.7%, 99.8%, 94.4%, 0.96 respectively) were higher than those of CEA (67.8%, 96.1%, 82.4%, 0.78 respectively) and VEGF (81.5%, 84.3%, 82.9%, 0.79 respectively). The sensitivity, specificity, accuracy and AUC(ROC) of IFN-gamma (85.7%, 96.4%, 90.9%, 0.94 respectively) were higher than those of ADA (80.2%, 87.6%, 83.8%, 0.81 respectively). It was concluded that VEGF/IFN-gamma ratio and IFN-gamma could be used as valuable parameters for the differential diagnosis of malignant pleural effusion and tuberculous pleural effusion.

Analysis of CD4+ CD25+ regulatory T cells and Foxp3 mRNA in the peripheral blood of patients with asthma.

The changes of CD4(+)CD25(+) regulatory T cells (CD4(+)CD25(+) Treg) and Foxp3 mRNA in peripheral blood mononuclear cells (PBMCs) from patients with asthma were investigated in order to elucidate the possible roles of CD4(+)CD25(+) Treg in the development of asthma. The peripheral blood samples were collected from 29 healthy controls (normal control group) and 78 patients with asthma which included 30 patients in exacerbation group, 25 patients in persistent group, and 23 patients in remission group. By using flow cytometry and RT-PCR, the CD4(+)CD25(+) Treg ratio and Foxp3 mRNA in PBMCs were detected. The CD4(+)CD25(+) Treg ratio and Foxp3 mRNA in PBMCs of exacerbation and persistent groups were lower than that of remission and normal control group (P<0.05). Although the CD4(+)CD25(+) Treg ratio and Foxp3 mRNA of remission group also lower than that of normal control group, there was no significant difference between them (P>0.05). As compared with persistent group, exacerbation group had lower CD4(+)CD25(+) Treg ratio and Foxp3 mRNA (P<0.05). It was indicated that the decrease of CD4(+)CD25(+) Treg ratio and its function in PBMCs may be responsible for pathogenesis of asthma.

Increased expression of PI-3K in asthmatic rat T lymphocytes.

In order to explore the expression of PI-3K in T lymphocytes of asthmatic rats and the relationship between PI-3K and activation of T lymphocytes, 24 Wistar rats were randomly divided into 4 groups: normal control group, asthmatic one-week group, asthmatic two-week group and asthmatic four-week group. T cells were purified from blood of each rat and the expression of PI-3K was observed by immunocytochemical fluorescence staining, the semiquantitative fluorescence intensity was measured by HPIAS-2000 analytic software, and the expression of IL-4 in supernatants was detected by ELISA. The results showed that the fluorescence intensity of T lymphocytes in asthmatic groups was significantly higher than that in normal control (P<0.001), indicating that the expression of PI-3K in T lymphocytes of asthmatic rats was significantly higher than that in those of normal controls, and the difference between acute and chronic stage asthmatic groups was significant (P<0.05). The expression levels of IL-4 protein in supernatants of asthmatic T lymphocytes were significantly higher than those in the normal controls (P<0.05). There was a significant positive correlation between the expression of PI-3K in T lymphocytes and the IL-4 protein expression in supernatants (r=0.583, P<0.01). It was suggested that PI-3K signal pathway may participate in the processes of activation and other cytological effects of asthmatic T lymphocytes, thus may play an important roles in the pathogenesis of asthma.