Biomarkers of some pulmonary diseases in exhaled breath

Analysis of various biomarkers in exhaled breath allows completely non-invasive monitoring of inflammation and oxidative stress in the respiratory tract in inflammatory lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), bronchiectasis and interstitial lung diseases. The technique is simple to perform, may be repeated frequently, and can be applied to children, including neonates, and patients with severe disease in whom more invasive procedures are not possible. Several volatile chemicals can be measured in the breath (nitric oxide, carbon monoxide, ammonia), and many non-volatile molecules (mediators, oxidation and nitration products, proteins) may be measured in exhaled breath condensate. Exhaled breath analysis may be used to quantify inflammation and oxidative stress in the respiratory tract, in differential diagnosis of airway disease and in the monitoring of therapy. Most progress has been made with exhaled nitric oxide (NO), which is increased in atopic asthma, is correlated with other inflammatory indices and is reduced by treatment with corticosteroids and antileukotrienes, but not (β₂-agonists. In contrast, exhaled NO is normal in COPD, reduced in CF and diagnostically low in primary ciliary dyskinesia. Exhaled carbon monoxide (CO) is increased in asthma, COPD and CF. Increased concentrations of 8-isoprostane, hydrogen peroxide, nitrite and 3-nitrotyrosine are found in exhaled breath condensate in inflammatory lung diseases. Furthermore, increased levels of lipid mediators are found in these diseases, with a differential pattern depending on the nature of the disease process. In the future it is likely that smaller and more sensitive analysers will extend the discriminatory value of exhaled breath analysis and that these techniques may be available to diagnose and monitor respiratory diseases in the general practice and home setting.     

Nitric oxide-modulated marker gene expression of signal transduction pathways in lung endothelial cells

Nitric oxide (NO) is a signal molecule involved in regulation of physiological and pathophysiological functions of the vascular endothelium such as apoptosis. We examined whether NO-modulates marker gene expression of signal transduction pathways in cultured pulmonary artery endothelial cell (PAEC). Cells were exposed to a NO donor, 1 mM NOC-18, for 0.5, 5, and 24 h, thereafter, expression levels of 96 marker genes associated with 18 signal transduction pathways were assessed using a signal transduction pathway-finder microarray analysis system. NO modulation of apoptotic pathways and nuclear factor (NF) microarray were further analyzed. Gene array analyses revealed that 17 genes in 13 signal pathways were up- or down-regulated in cells exposed to NO, four of which were significantly altered by NO and are associated with apoptotic pathways. Apoptotic pathways resulted in identification of 11 genes in this group. Nuclear factor microarray studies demonstrated that NO-modulated expression of these signal transduction genes was associated with regulation of NF-binding activities. Gel shift analysis verified the effects of NO on DNA-binding activity of NF. These results demonstrated that NO signaling modulates at least 13 signal transduction pathways including apoptosis-related families in PAEC.     

胎肺细胞肾包膜下种植模型--胎肺发育研究之新体内模型

肺是哺乳动物重要的呼吸器官,其发育的大部分过程发生于胚胎阶段,但由于研究手段的限制,对胎肺特别是后期胎肺发生机制的认识还十分有限.本文利用肾包膜下种植的方法建立了胎肺细胞肾包膜下种植模型.模型中上皮发育历经假腺体期、小管期和肺泡前体期等正常胎肺上皮组织发育的所有分化阶段,同时间充质形成广泛的毛细血管网络,与胎肺在子宫中的发育过程一致.更重要的是,消化处理后的单个胎肺细胞可有效地吸收反义寡核苷酸,并在种植组织中产生相应的表型效应.模型的建立为胎肺发生机制研究提供了一条新的途径.     

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-α in lung cancer

INTRODUCTION: Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. RESULTS: Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. CONCLUSION: These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.     

TTF-1和HBME-1在肺癌患者胸水中的表达及意义

目的探讨TTF-1和HBME-1在肺癌患者胸水中的诊断价值。方法应用免疫细胞化学方法（S-P）研究51例原发性肺癌患者和10例继发性肺癌患者胸水中的癌细胞及44例肺良性疾病胸水中反应性间皮细胞的表达。结果TTF-1在原发性肺癌患者胸水中的阳性率为78.4%（40/51）,而在继发性肺癌患者和肺良性疾病患者的胸水中未见阳性表达;HBME-1在肺良性疾病患者胸水中的阳性率95.5%（42/44）明显高于在原发性肺癌患者胸水中25.5%（13/51）和继发性肺癌患者胸水的阳性率20.0%（2/10）;在不同病理类型原发性肺癌患者胸水中,TTF-1的阳性率在腺癌组90.7%（39/43）明显高于鳞癌组12.5%（1/8）;当TTF-1和HBME-1联合应用时为最佳选择,其检测肺癌患者胸水的敏感性和特异性分别高达96.7%和95.5%。结论TTF-1和HBME-1对原发性、继发性肺癌患者胸水中癌细胞的诊断及鉴别诊断具有重要的临床应用价值。     

Haplotypes of nine single nucleotide polymorphisms on chromosome 19q13.2-3 associated with susceptibility of lung cancer in a Chinese population

To evaluate the joint effect of nine single nucleotide polymorphisms for three DNA repair genes in the region of chromosome 19q13.2-3 on susceptibility of lung cancer in a Chinese population, we conducted a hospital-based case–control study consisting of 247 lung cancer cases and 253 cancer-free controls matched on age, gender and ethnicity. Associations between the haplotypes and susceptibility of lung cancer were tested. The global test of haplotype association revealed a statistically significant difference in the haplotype distribution between cases and controls (global test: χ² = 60.45, d.f. = 15, P = 2.11E−07). The two haplotypes were underrepresented among cases (Hap5 defined by ERCC1118^A–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^T–XRCC1206^A–XRCC1280^G–XRCC1399^G–XRCC1632^G and Hap12 defined by ERCC1118^G–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^C–XRCC1206^A–XRCC1280^G–XRCC1399^A–XRCC1632^G). Three of the haplotypes were overrepresented among cases (Hap3 defined by ERCC1118^A–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^C–XRCC1206^A–XRCC1280^G–XRCC1399^G–XRCC1632^G, Hap4 defined by ERCC1118^A–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^C–XRCC1206^G–XRCC1280^G–XRCC1399^G–XRCC1632^A, and Hap10 defined by ERCC1118^G–ERCC2156^A–ERCC2312^G–ERCC2751^A–XRCC1194^T–XRCC1206^A–XRCC1280^G–XRCC1399^G–XRCC1632^G). Haplotypes 3 and 10 (cases = 5.7%, controls = 1.0%, OR = 6.56, 95%CI = 1.83–23.54, P = 0.001; cases = 13.3%, controls = 5.6%, OR = 2.73, 95%CI = 1.51–4.94, P = 0.0006) were the most strongly associated with increased lung cancer risk. There was considerable linkage disequilibrium exists between SNPs both within genes and between genes in the region. The two blocks for solid spine of LD and six htSNPs were found. The haplotype analysis suggested that the biologically effective polymorphisms co-segregate with some of the haplotypes. This result supports the hypothesis that the sub-region is important for lung cancer susceptibility. Haplotype studies using larger study groups will be required to obtain conclusive results.     

Halothane-induced alterations in cellular structure and proliferation of A549 cells

Genotoxicity, cytotoxicity or teratogenicity are among the well-known detrimental effects of the volatile anaesthetics. The aim of the present work was to study the structural changes, proliferative activity and the possibility of alveolar A549 cells to recover after in vitro exposure to halothane at 1.5 and 2.1 mM concentrations. Our data indicated significant reduction of viability, suppression of mitotic activity more than 60%, and that these alterations were accompanied by disturbances of nuclear and nucleolar structures. The most prominent negative effect was the destruction of the lamellar bodies, the main storage organelles of pulmonary surfactant, substantial for the lung physiology. In conclusion, halothane applied at clinically relevant concentrations exerts genotoxic and cytotoxic effect on the alveolar cells in vitro, most likely as a consequence of stress-induced apoptosis, thus modulating the respiratory function.     

Helicobacter pylori lipopolysaccharide provokes iNOS-mediated acute systemic microvascular inflammatory responses in rat cardiac, hepatic, renal and pulmonary tissues

We have examined the effects of intravenous administration of a purified lipopolysaccharide (LPS) from Helicobacter pylori (3 mg kg⁻¹, i.v.) on rat vascular permeability, assessed by the radiolabelled human serum albumin leakage technique in the heart, kidney, liver and lung 4 h after challenge. An increased vascular permeability in cardiac, renal, hepatic and pulmonary tissues after challenge was determined. The albumin leakage observed in all these organs could be prevented by the selective inducible nitric oxide synthase inhibitor, N-(8-(aminomethyl)benzyl)-acetamidine (1400W; 0.2–1 mg kg⁻¹, s.c.) administered concurrently with LPS. Thus, H. pylori LPS can provoke a microvascular inflammatory response in the rat cardiac, renal, hepatic and pulmonary tissues, actions mediated through the activation of the inducible nitric oxide synhase isoenzyme.     

Dual Y chromosome painting and in situ cell-specific immunofluorescence staining in lung tissue: an improved method of identifying donor marrow cells in lung following bone marrow transplantation

Several recent studies have demonstrated localization of donor bone marrow-derived cells in recipient lungs following transplantation from male to female mice or patients. Donor cells are identified by detection of the Y chromosome by fluorescence in situ hybridization (FISH). However, protein digestion pretreatments usually required for tissue FISH significantly limit the ability to detect cell type-specific markers by immunohistochemistry. We have used an alternative protein digest approach that entails heating the slides in 10 mM sodium citrate rather than utilizing a protease digestion. This approach preserves cell proteins following FISH, and allows lung tissue to remain intact for subsequent detection of cell-specific markers by immunohistochemistry. We have examined this technique in mouse lungs using a Y chromosome paint and three cell-specific markers, a pan-cytokeratin for epithelial cells, PECAM-1 for endothelial cells, and CD45 for leukocytes. Excellent visualization of both the Y chromosome and cell-specific surface protein markers was obtained on a single slide. This approach will significantly enhance the ability to detect and identify donor bone marrow cells in recipient mouse lungs following male to female transplantation.