Objective To investigate clinical significance of small airway function and eosinophil (Eos) percentage,levels of eosinophil cationic protein (ECP) and IL-5 in induced sputum among patients with clinically controlled asthma. Methods Sixty-two patients with clinically controlled asthma were selected for the study. Lung function was performed and percentage of Eos, levels of ECP and IL-5 in induced sputum were measured by Wrights' stain, fluorescence immuno-CAP system and ELISA,respectively. Thirty patients of asthma at acute exacerbation period and 20 healthy subjects were selected as controls. Results In 62 patients with clinically controlled asthma, 43 (69. 4% ) showed abnormal small airway function and 19(30. 6% ) normal one. Percentage of Eos [(5. 6 ±2. 9)%], levels of ECP [( 129 ±100) μg/L] and IL-5 [(21± 12) μg/L] in induced sputum were significantly lower in patients with clinically controlled asthma than those of asthma at acute exacerbation period [( 16. 2 ± 9. 7 ) %, ( 362 ±182) μg/L and IL-5(51 ±26) μg/L, respectively] (all P ＜0. 01 ), but significantly higher than those in healthy controls ( all P ＜ 0. 01 ). Percentage of Eos, levels of ECP and IL-5 in induced sputum were significantly higher in patients with clinically controlled asthma with abnormal small airway function than those with normal ane [(6.9±3.1)% vs. (2.0±1.1)%, (148±90) μg/Lvs. (54±29) μg/L and (24 ±12) μg/L vs. ( 13 ± 5 ) μg/L, respectively] ( all P ＜ 0. 01 ). Conclusions Abnormal small airway function and airway inflammation persistently exist in patients with clinically controlled asthma and it may be helpful to guild treatment during clinical control to determine small airway function and inflammatory markers in their induced sputum.

BACKGROUND: The different level of proteins regulating cell cycle and theircorrelation is the main criteria to differentiate the benign and malignant cellular proliferation.OBJECTIVE: To investigate the expression status of p21waf1 and p53 in lung cancer as well as proliferating cell nuclear antigen (PCNA)DESIGN:A case-control study.SETTING: Department of Respiratory Medicine, Renmin Hospital ,Wuhan UniversityPAITICIPANTS:This case-control study involved 135 patients who underwent lobectomy or fiberoptic bronchoscopy for primary lung cancer or benign chronic pulmonary diseases at Renmin Hospital of Wuhan University from October 1996 through May 1999. They were divided into two groups: lung cancer group (76 patients, including 56 men and 20 women,aged 18-74 years of age) and chronic pulmonary diseases group (59 cases,including 42 men and 17 women, aged 16-70 years of age).METHODS: Phosphate buffer solution replaced the first antibody as the negative control. Immunohistochemistry was performed using a modified streptavidin-biotinylated peroxidase technique according to the manufacturer's recommendations (Maxim Corporation). For p21waf1 staining, we used hydrated autoclaving as a pretreatment. Antigen retrieval was performed in a standard microwave unit for p53 staining. PCNA staining did not need The ratio of the positive cells indicated by yellowish brown nucleus due to staining was counted for 5 successive high-fold microscopic fields: when it was≥ 10%, it was taken as positive; when it was ＜10%, it was regarded as high-fold microscopic fields for the percentage of the positive cells indicated by yellowish brown nucleus due to staining in each field, and the average value of the five fields was taken as labeling index (LI) for proliferated nuclear antigens.MAIN OUTCOME MEASURES: The expression leyels of p21waf1, p53and PCNA in lung cancer.cancer were 75% (57/76) and 47%(36/76) respectively. The labeling index of PCNA in lung cancer group was significantly higher than that of the chronic pulmonary diseases group (0.44±0.32 vs 0.09±0.14, respectively).significantly higher than that of small cell lung cancer (0.51 ±0.33 vs in lung cancer tissues. In chronic pulmonary diseases group, the expression of p21waf1 and p53 showed a close relationship with PCNA.CONCLUSION: It was found that p21waf1 and p53 were obviously upregulated in lung cancer and the degree of cellular proliferation in lung cancer was rather high. The capacity of DNA damage repair in squamous lung cancer may be stronger than that in small cell lung cancer.

Objective To determine the effect of Vibrio vulnificus cytolysin (VVC) inducing ap-optosis in human umbilical vein endothelial cell(HUVEC) and its possible mechanism. Methods The en-tire vvhA gene that encoding VVC from V. vulnificus strain GTC333 was amplified by PCR and sequenced af-ter T-A cloning. E. coli BL21DE3pET-42a-vvhA, a prokaryotic expression system of the vvhA gene, was then con-structed. Ni-NTA affinity chromatography was applied to purify the target recombinant protein rVVC, and SDS-PAGE plus Bio-Rad Agarose Image Analyzor were used to measure the output of rVVC and to determine the purity of rVVC extract. The activity of rVVC dissolving rabbit erythrocytes was detected by hemolysis test. DPNH chromotometry and TphBNa chromotometry were performed to examine the contents of LDH and K+ in the supernatants of rVVC-treated HUVEC cultures, respectively. The effect of rVVC inducing apepto-sis of HUVEC was detected by flow cytometry, rVVC was labeled with FITC and the location of FITC-labe-ling rVVC in HUVEC was observed by laser canfocal microscopy. Results The cloned whA gene had 96.09% and 98.26% similarities of nucleotide and amino acid sequences compared to the corresponding se-quences in GenBank. rVVC, with a dosage of 1 μg/ml, could dissolve rabbit erythrocytes (P<0.01). 10 μg/ml rVVC was able to promote the increases of K+ content (P<0.01) but no change of LDH content could be found in the cell supernatants. HUVEC was apoptotic after the cell was treated with 1～100 μg/ml of rVVC for 2 h. In the 5～240 min duration of co-incubation of FITC-labeling rVVC and HUVEC, the rV-VC gradually moved from surface to inner side of the membrane and then entered the cytoplasms. When FITC-labeling rVVC treated HUVEC for 30 min, most of the rVVC was found to be intracellular location. Conclusion rVVC has cytolytic activity. VVC has an ability to enter HUVEC and causes injury of HUVEC via inducing apoptosis, which may be the major pathogenic mechanism of VVC.

Plasma level of N-terminal pro brain natriuretic peptide (NT-proBNP) was measured for 362 elderly patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD), including 97 of AECOPD complicated with left-heart failure and 265 of isolated AECOPD.Results indicated that there was significant difference in plasma level of NT-proBNP between the two groups ( P = 0.000).With a cut-off value of 1643.5 ng/L NT-proBNP, its sensitivity, specificity and accuracy for identifying AECOPD complicated with left-heart failure in the elderly were 84.4%, 85.3% and 85.1%, respectively.It is suggested that assay for plasma NT-proBNP may be helpful to identify left-heart failure in elderly patients with AECOPD.

To explore the clinical significance of changes in maximal expiratory flow in 50％ and 25％ vital capacity (Vmax50％ & Vmax25％) before and after bronchodilator reversibility testing in patients with asthma.Forced expiratory volume in one second (FEV1),Vmax50％ and Vmax25％ were measured before and after bronchodilator reversibility testing in 118 patients with asthma and 82 with chronic obstructive pulmonary disease (COPD).The rate of positive reversibility in Vmax50％ was significantly higher than that in FEV1 in 118 asthmatics (x2 =7.995,P =0.007).The rates of positive reversibility in Vmax50％ and Vmax25％ were significantly higher in asthmatics than those in COPD patients (x2 =9.335,P =0.009).

Objective:To investigate the effect of type ⅡNKT cells activated by sulfatide on airway inflammation in a murine model of asthma.Methods:Thirty-two BALB/c mice were randomly divided into four groups:normal control group ( n=8 ) , asthma group (n=8),sulfatide treatment group (n=8) and adoptive transfer group (n=8).The murine model of asthma was established by sensitization with intraperitoneal injection of ovalbumin ( OVA) and intranasal challenge in all animals except for the normal control group where PBS was used instead.Intraperitoneal injection of sulfatide in a sulfatide treatment group, adoptive transfer of sulfatide-activated typeⅡNKT cells in adoptive transfer group and PBS in asthma group were carried out 1 hour before the first challenge.PBS was used for intraperitoneal administration in the normal control group.Lung histology and goblet cell hyperplasia were analyzed by HE or PAS staining.Differential cell count in bronchial alveolar lavage ( BALF) was measured by May-Gruenwald Giemsa;levels of OVA-specific IgE in serum and L-4,IL-5 in BALF were measured by ELISA.The percentages of lung type Ⅱ NKT cells,IL-4+and IFN-γ+typeⅡNKT cells were detected by flow cytometry.Results:Inflammatory cell infiltration in lung tissue and goblet cell hyperplasia in the airway were decreased in sulfatide treatment group and adoptive transfer group.Percentages of eosinophil in BALF,level of OVA-specific IgE in serum,and levels of IL-4,IL-5 in BALF in sulfatide treatment group and adoptive transfer group were significantly lower than those in asthma group (all P<0.05).The percentages of lung IL-4+and IFN-γ+typeⅡNKT cells in sulfatide treatment group was significantly higher than those in asthma group ( P<0.01 ).Conclusion: Type Ⅱ NKT cells activated by sulfatide may inhibit airway inflammation in a murine model of asthma.

Objective To explore the effects of theophylline plus salmeterol/fluticasone propionate combination product (SFC) on clinical control,lung function and airway inflammation in asthmatics.Methods A total of 146 asthmatics received 200 mg theophylline plus 50/250 μg SFC twice daily for 24 weeks.The level of asthma control was assessed by the asthma control test.Testing of lung function and inflammatory markers in induced sputum were performed.And 142 asthmatics received 50/250 μg SFC twice daily for 24 weeks as control.Results Asthma was completely controlled in 61 and 59 in the theophylline plus SFC and SFC groups respectively after a 24-week treatment period (P ＞ 0.05).Theophylline plus SFC improved the FEF25％ 75％ value,indicating small airway function,to a greater extent than SFC [(66.7 ± 18.2) ％ & (56.6 ± 17.4) ％,P ＜ 0.01].Percentage of eosinophil and concentration of eosinophil cationic protein in induced sputum were significantly lower in the theophylline plus SFC group than those in the SFC group [(4.1 ±2.3)％ vs.(6.2±2.7)％ & (63.9±39.4) vs.(90.3 ±46.2) μg/Lrespeetively] (all P ＜ 0.01).Conclusion The therapy of theophylline plus SFC may provide greater improvements in small airway function and airway inflammation.

OBJECTIVETo study the molecular pathogenesis of protein C (PC) deficiency in a patient with pulmonary embolism and in his family members.

METHODSAnticoagulated blood samples were collected from the proband and his family members to detect PC, PS and AT activities. PC antigen level was measured using ELISA. The genomic DNA was extracted to amplify all the 9 exons and their flanking sequences of PC gene using PCR, and the PCR products were sequenced. The mutated exons identified were amplified and sequenced for the other family members.

RESULTSThe proband and his parents and sister were identified as carriers of PC gene mutation, which led to type II PC deficiency. Sequencing of the proband's PC gene showed two heterozygous point mutations in exon 3 (G5540A) and exon 7 (C10230T) to cause compound heterozygous mutations of PC E29K and PC R147W, which were inherited from his father and mother, respectively. His sister was a heterozygote of PC R147W.

CONCLUSIONThe proband is a compourd heterozygous mutations carrier of PC E29K and PC147W. PC E29K is a novel PC mutation, and PC R147W is a reported PC gene mutation seen in patients with type II hereditary PC deficiency and recurrent thrombosis.

Adolescent ; Base Sequence ; Heterozygote ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Point Mutation ; Protein C ; genetics ; Protein C Deficiency ; complications ; genetics ; pathology ; Pulmonary Embolism ; etiology ; genetics

Objective:To observe the antibacterial effect of Ag +-loaded TiO 2 (Ag -TiO 2) and Ag -TiO 2 coated endotracheal tube (ETT) on the bacterial biofilm (BF) of Staphylococcus aureus. Methods:2, 3-bis-(2-methoxy- 4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric method was used to detect minimal inhibitory concertation (MIC) of Ag-TiO 2 for inhibition of BF of Staphylococcus aureus. The Ag -TiO 2 coated ETT were prepared, and divided into 11 mg/L, 8 mg/L, 5 mg/L, 2 mg/L and 0 mg/L ETT group, according to the concentration gradient, then impregnated in the liquid with Staphylococcus aureus at a concentration of 1.0×10 9cfu/L. The influence of antibacterial coated ETT on the formation of Staphylococcus aureus BF was determined by detecting the colonies of bacteria and BF on the ETT. Results:Ag-TiO 2 had a significant inhibitory effect on Staphylococcus aureus BF in a concentration -dependent manner, and its MIC was 10 mg/L. Ag -TiO 2 coated ETT has significant anti -Staphylococcus aureus BF effect, and the higher the concentration, the stronger the effect. The absorbance ( A) values of Ag -TiO 2 5 mg/L, 8 mg/L, 11 mg/L ETT groups were significantly lower than that in control group (0.176±0.004, 0.147±0.002, 0.094±0.002 vs. 0.267±0.045, all P < 0.05). The inhibitory rates of Ag -TiO 2 2 mg/L, 5 mg/L, 8 mg/L ETT groups were increased gradually, and 11 mg/L Ag -TiO 2 coated ETT group had the highest inhibitory rate for BF, the inhibitory rates were 6.4%, 34.1%, 44.9% and 64.8%, respectively. Conclusion:Both Ag-TiO 2 and Ag-TiO 2 coated ETT have significant inhibitory effects on Staphylococcus aureus BF.

Objective:To confirm the Sigma N transcription factor activity of a gene product encoded by LA2404 gene of Leptospira interrogans ( L. interrogans) and to identify the target genes of Sigma N signaling system. Methods:L. interrogans LA2404 gene and its regulated target genes were predicted using bioinformatic analysis according to the promoter sequence signature in Sigma N-regulated genes. A LA2404 gene-knockout (ΔLA2404) strain of L. interrogans was constructed based on homologous sequence recombinant of suicide plasmid. Real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to detect the changes in the expression of target genes at mRNA level in the ΔLA2404 mutant. A prokaryotic expression system for LA2404 gene was established and the target recombinant protein rSigma N was extracted by Ni-NTA affinity chromatography. Gel electrophoresis mobility shift assay (EMSA) was used to screen out the target genes regulated by rSigma N. Results:Pathogenic L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai carried one Sigma N gene and 22 Sigma N promoter sequence-containing target genes. Qualitative examination of the ΔLA2404 mutant by microscopy revealed no defect in motility and appearance. Expression of LA1188, LA2306, LA3426, LA1968, LA1313, LA3806 and LA0773 genes at mRNA level in the ΔLA2404 mutant was significantly down-regulated ( P<0.05), but no significant changes in the expression of other target genes at mRNA level were detected. EMSA results confirmed that rSigma N could bind to the promotor sequences of the target genes mentioned above. Conclusions:Sigma N transcription factor was encoded by LA2404 gene. LA1188, LA2306, LA3426, LA1968, LA1313, LA3806 and LA0773 genes contained Sigma N promoter sequence and the expression of them was regulated by Sigma N signaling system.