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1.  PCR based bronchoscopic detection of common respiratory pathogens in chronic cough: a case control study 
Background
Viral respiratory tract infection is the most frequent cause of acute cough and is reported at onset in about one third of patients with chronic cough. Persistent infection is therefore one possible explanation for the cough reflex hypersensitivity and pulmonary inflammation reported in chronic cough patients.
Methods
Bronchoscopic endobronchial biopsies and bronchoalveolar lavage cell counts were obtained from ten healthy volunteers and twenty treatment resistant chronic cough patients (10 selected for lavage lymphocytosis). A screen for known respiratory pathogens was performed on biopsy tissue. Chronic cough patients also underwent cough reflex sensitivity testing using citric acid.
Results
There was no significant difference in incidence of infection between healthy volunteers and chronic cough patients (p = 0.115) or non-lymphocytic and lymphocytic groups (p = 0.404). BAL cell percentages were not significantly different between healthy volunteers and chronic cough patients without lymphocytosis. Lymphocytic patients however had a significantly raised percentage of lymphocytes (p < 0.01), neutrophils (p < 0.05), eosinophils (p < 0.05) and decreased macrophages (p < 0.001) verses healthy volunteers. There was no significant difference in the cough reflex sensitivity between non-lymphocytic and lymphocytic patients (p = 0.536).
Conclusions
This study indicates latent infection in the lung is unlikely to play an important role in chronic cough, but a role for undetected or undetectable pathogens in either the lung or a distal site could not be ruled out.
Trials registration
Current Controlled Trials ISRCTN62337037 & ISRCTN40147207
doi:10.1186/1745-9974-8-5
PMCID: PMC3496690  PMID: 22978556
Biopsy; Bronchoalveolar lavage; Bronchoscopy; Cough; Infection; PCR; Virus
2.  Characterizing Mucous Cell Remodeling in Cystic Fibrosis 
Rationale: Relatively few studies have characterized mucous cells or mucins in detail in cystic fibrosis (CF), and the relationship between mucous cell abnormalities and neutrophilic inflammation is uncertain.
Objectives: To characterize mucous cell phenotypes and mucin profiles in CF and to determine if neutrophils accumulate around goblet cells in the epithelium and gland acini in the submucosa.
Methods: Bronchial biopsies were collected from 7 subjects with CF and 15 control subjects, and the morphology of mucous cells was measured. Immunostains for gel-forming mucins and neutrophil elastase were quantified.
Measurements and Main Results: Goblet cell size was increased in CF (p = 0.004), but the number of goblet cells was normal. The volume of submucosal glands was fourfold higher than normal (p = 0.031), but the proportion of mucous and serous cells in CF glands was normal. The patterns of expression of gel-forming mucins in epithelial and submucosal compartments in CF were similar to normal. Although neutrophil elastase immunostaining was intense in the epithelium in CF, neutrophils were largely absent around gland acini in the submucosa.
Conclusion: The most prominent pathologic feature in the CF airway is an increase in submucosal gland volume, but serous cell transdifferentiation to mucous cells does not occur, nor are gland acini inflamed with neutrophils. The mechanism for increased submucosal gland volume in CF deserves further study.
doi:10.1164/rccm.200603-310OC
PMCID: PMC2648101  PMID: 16917116
cystic fibrosis; MUC5AC; MUC5B; neutrophil elastase; submucosal glands
3.  Response to Acute Lung Infection with Mucoid Pseudomonas aeruginosa in Cystic Fibrosis Mice 
Rationale: Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator gene, which codes for a chloride channel, but the role of this chloride channel in inflammation induced by lung infection with Pseudomonas aeruginosa remains to be defined.
Objectives: We tested the hypothesis that loss of this chloride channel alone is sufficient to cause excessive inflammation in response to inflammatory stimuli.
Methods: We investigated the response of cystic fibrosis and wild-type mice to mucoid P. aeruginosa administered by insufflation.
Measurements: The host responses measured included survival, weight change, lung morphometry, bacterial clearance, and inflammatory mediators, and cell counts were assessed in bronchoalveolar lavage fluid.
Main Results: Depending on the dose administered and frequency of dosing, cystic fibrosis mice experienced significantly higher mortality rates, greater weight loss, higher lung pathology scores, and higher inflammatory mediator and neutrophil levels compared with wild-type mice, even after the bacteria had been cleared. Surprisingly, bacteria were cleared just as rapidly in cystic fibrosis mice as in wild-type mice, and sepsis was not observed. Chronic lung infections could not be established with mucoid P. aeruginosa in either cystic fibrosis or wild-type mice.
Conclusions: Absence of this chloride channel alone appears sufficient for exaggerated inflammation and excess mortality compared with wild-type controls in the face of mucoid P. aeruginosa lung infection. To establish chronic infection, additional factors such as bacterial trapping or poor clearance may be required.
doi:10.1164/rccm.200506-917OC
PMCID: PMC2662931  PMID: 16272448
cystic fibrosis; cystic fibrosis transmembrane conductance regulator; inflammation; pathogenesis
4.  Activated memory T helper cells in bronchoalveolar lavage fluid from patients with atopic asthma: relation to asthma symptoms, lung function, and bronchial responsiveness. 
Thorax  1993;48(1):26-32.
BACKGROUND: Bronchial mucosal inflammation and epithelial damage are characteristic features of asthma. Activation of T helper lymphocytes may contribute to this process by mechanisms including the release of cytokines promoting eosinophil infiltration and activation. METHODS: Bronchial washings and bronchoalveolar lavage fluid were obtained from 29 atopic asthmatic patients (19 with current symptoms and 10 symptom free) and 13 normal volunteers. Flow cytometry was used to assess T cell phenotype and activation status in bronchoalveolar lavage fluid and peripheral blood, and differential cell counts were made on bronchial washings and bronchoalveolar lavage fluid. Findings were related to severity of disease as reflected by symptom scores, baseline lung function, and airway responsiveness. RESULTS: CD4 T lymphocytes in bronchoalveolar lavage fluid and blood from asthmatic patients were activated by comparison with controls (CD4 CD25, median 16.8% v 8.7% for bronchoalveolar lavage fluid, and 15.3% v 8.7% in blood). Bronchoalveolar lavage fluid CD4 T cells from both asthmatic patients and controls were of memory phenotype (95.8% and 96.8% CD45RO and 1.7% and 0.4% CD45RA respectively), whereas both CD45RO and CD45RA T cells were present in blood. Patients with asthma and current symptoms showed increased bronchoalveolar T cell activation compared with patients without symptoms (CD4 CD25 18.7% v 12.3%). Within the asthmatic group there was a significant association between CD4 CD25 lymphocytes and asthma symptom scores (rs = 0.75), airway methacholine responsiveness (log PC20, rs = -0.43) and baseline FEV1 (rs = -0.39). A correlation was also found between CD4 CD25 lymphocytes and eosinophils in bronchoalveolar lavage fluid (rs = 0.48). Eosinophils in bronchoalveolar lavage fluid were increased in asthmatic patients compared with controls and the percentage of eosinophils in bronchoalveolar lavage fluid correlated with asthma symptom score. A relation was found between percentage of epithelial cells in bronchoalveolar lavage fluid and FEV1 and methacholine PC20. CONCLUSION: These results support the hypothesis that selective activation of memory CD4 T cells contributes to eosinophil accumulation, bronchial hyperresponsiveness, and symptoms in asthma.
PMCID: PMC464232  PMID: 8434349
5.  Airway remodelling in children with cystic fibrosis 
Thorax  2007;62(12):1074-1080.
Background
The relationship between airway structural changes and inflammation is unclear in early cystic fibrosis (CF) lung disease. A study was undertaken to determine changes in airway remodelling in children with CF compared with appropriate disease and healthy controls.
Methods
Bronchoalveolar lavage and endobronchial biopsy were performed in a cross‐sectional study of 43 children with CF (aged 0.3–16.8 years), 7 children with primary ciliary dyskinesia (PCD), 26 with chronic respiratory symptoms (CRS) investigated for recurrent infection and/or cough and 7 control children with no lower airway symptoms. Inflammatory cells, cytokines, proteases and matrix constituents were measured in bronchoalveolar lavage fluid (BALF). Reticular basement membrane (RBM) thickness was measured on biopsy specimens using light microscopy.
Results
Increased concentrations of elastin, glycosaminoglycans and collagen were found in BALF from children with CF compared with the CRS group and controls, each correlating positively with age, neutrophil count and proteases (elastase activity and matrix metalloproteinase‐9 (MMP‐9) concentration). There were significant negative correlations between certain of these and pulmonary function (forced expiratory volume in 1 s) in the CF group (elastin: r = −0.45, p<0.05; MMP‐9:TIMP‐1 ratio: r = −0.47, p<0.05). Median RBM thickness was greater in the CF group than in the controls (5.9 μm vs 4.0 μm, p<0.01) and correlated positively with levels of transforming growth factor‐β1 (TGF‐β1; r = 0.53, p = 0.01), although not with other inflammatory markers or pulmonary function.
Conclusions
This study provides evidence for two forms of airway remodelling in children with CF: (1) matrix breakdown, related to inflammation, proteolysis and impaired pulmonary function, and (2) RBM thickening, related to TGF‐β1 concentration but independent of other markers of inflammation.
doi:10.1136/thx.2006.074641
PMCID: PMC2094274  PMID: 17526676
6.  The immunological component of the cellular inflammatory infiltrate in bronchiectasis. 
Thorax  1989;44(8):668-673.
Immunohistological analysis of bronchial biopsy specimens from nine patients with bronchiectasis and four control subjects was performed with a panel of monoclonal antibodies selected to show lymphocyte and macrophage subsets and signs of cellular activation. The cells taking part in the inflammatory response in the bronchial wall of patients with bronchiectasis were almost exclusively mononuclear cells, most of them T lymphocytes. B lymphocytes were observed in biopsy specimens from only two out of nine patients. CD8+ T cells outnumbered CD4+ cells in all patients in a ratio ranging from 2:1 to 10:1. Most T lymphocytes also strongly expressed CD7 antigen and a proportion of them expressed HLA-DR. Most of the lymphocytic infiltration occurred just beneath the basement membrane of the epithelium, though intraepithelial and submucosal infiltration was also seen. Non-lymphoid mononuclear cells expressing the phenotype of dendritic cells and macrophages were found dispersed throughout the infiltrate, most of them expressing HLA-DR. These observations support the hypothesis that cell mediated immunological reactions contribute to the inflammation associated with bronchiectasis.
Images
PMCID: PMC462004  PMID: 2678579
7.  Chronic lymphocytic leukaemia, dyspnoea and “tree-in-bud” sign on chest CT scan 
BMJ Case Reports  2009;2009:bcr03.2009.1672.
Chronic lymphocytic leukaemia (CLL) is a common disorder. Patients typically present with lymphadenopathy, splenomegaly and marked lymphocytosis (often >100 000/μl). Although pulmonary involvement from CLL can be found in more than one third of patients on autopsy, respiratory symptoms caused by the disease itself are not often reported. Pulmonary involvement mainly includes parenchymal infiltrates, peribronchial and perivascular infiltration, recurrent bacterial pneumonia, oedema or infarction, pleural effusions, and lymphadenopathy. Occasionally, patients may present with dry cough and progressive dyspnoea, even with low peripheral white blood cell count. We report a case of CLL and dyspnoea at rest, predominant “tree-in-bud” sign on chest computed tomography scan, and biopsy proven bronchiolar infiltration with monoclonal lymphocytes. With bronchoalveolar lavage alone, the diagnosis would have been missed. Chemotherapy with rituximab, cyclophosphamide, and fludarabinphosphate led to a prompt clinical and radiological improvement with a gain in 6 min walking distance from 60 to 210 m.
doi:10.1136/bcr.03.2009.1672
PMCID: PMC3027947  PMID: 21853008
8.  CD8+ T lymphocytes in bronchoalveolar lavage in idiopathic pulmonary fibrosis 
Background
Recently it was shown that in Idiopathic Pulmonary Fibrosis (IPF) tissue infiltrating CD8+ T lymphocytes (TLs) are associated with breathlessness and physiological indices of disease severity, as well as that CD8+ TLs recovered by bronchoalveolar lavage (BAL) relate to those infiltrating lung tissue. Since BAL is a far less invasive technique than tissue biopsy to study mechanisms in IPF we further investigated the usefulness offered by this means by studying the relationship between BAL macrophages, neutrophils, eosinophils, CD3+, CD4+, CD8+, CD8+/38+ TLs and CD4+/CD8+ ratio with breathlessness and physiological indices.
Patients and methods
27 IPF patients, 63 ± 9 years of age were examined. Cell counts were expressed as percentages of total cells and TLs were evaluated by flow cytometry. FEV1, FVC, TLC, RV, DLCO, PaO2, and PaCO2 were measured in all. Breathlessness was assessed by the Medical Research Council (MRC) chronic dyspnoea scale.
Results
CD8+ TLs correlated positively (rs = 0.46, p = 0.02), while CD4+/CD8+ ratio negatively (rs = -0.54, p = 0.006) with the MRC grade. CD8+ TLs correlated negatively with RV (rs = -0.50, p = 0.017). CD8+/38+ TLs were negatively related to the FEV1 and FVC (rs = -0.53, p = 0.03 and rs = -0.59, p = 0.02, respectively). Neutrophils correlated positively with the MRC grade (rs = 0.42, p = 0.03), and negatively with the DLCO (rs = -0.54, p = 0.005), PaO2 (rs = -0.44, p = 0.03), and PaCO2 (rs = -0.52, p = 0.01).
Conclusion
BAL CD8+ TLs associations with physiological and clinical indices seem to indicate their implication in IPF pathogenesis, confirming our previous tissue study.
doi:10.1186/1476-9255-4-14
PMCID: PMC1906752  PMID: 17578573
9.  Correlation of CD4:CD8 ratio and tumour necrosis factor (TNF)α levels in induced sputum with bronchoalveolar lavage fluid in pulmonary sarcoidosis 
Thorax  2000;55(8):696-699.
BACKGROUND—An increased CD4:CD8 lymphocyte ratio and raised cytokine levels in bronchoalveolar lavage (BAL) fluid are characteristic of pulmonary sarcoidosis. Sputum induction has been used as a non-invasive tool for investigating the airways and may be useful in investigating inflammation in patients with sarcoidosis in whom endobronchial, peribronchial, and parenchymal inflammation is present. This study aimed to correlate the total and differential cell counts, CD4:CD8 ratio, and tumour necrosis factor (TNF)α levels between induced sputum and BAL fluid in patients with pulmonary sarcoidosis.
METHODS—Fourteen patients with newly diagnosed biopsy proven sarcoidosis and six healthy controls were investigated. Sputum induction and BAL was carried out at the initial visit and repeated following six months of treatment with oral prednisone.
RESULTS—There was no correlation of differential cell counts between induced sputum and BAL fluid. The CD4:CD8 ratio in induced sputum correlated strongly with that in BAL fluid (5.5 (0.4:1) versus 4.4 (0.2:1); r = 0.8, p<0.001) and the fall in the ratio following six months of treatment in sputum paralleled that in BAL fluid (3.4 (0.2:1) versus 2.4 (0.2:1)). The TNFα levels in sputum also correlated with levels in the BAL fluid (11.9 (1.5) pg/ml versus 17.6 (2.7) pg/ml; r = 0.8, p<0.001). The fall in sputum TNFα levels following six months of treatment paralleled the fall in BAL fluid levels (6.7 (0.9) pg/ml versus 11.6 (1.3) pg/ml).
CONCLUSIONS—The CD4:CD8 ratio and TNFα levels in induced sputum correlated with those in BAL fluid and paralleled changes with treatment. Induced sputum may therefore be a non-invasive surrogate for certain parameters in BAL fluid in patients with sarcoidosis.


doi:10.1136/thorax.55.8.696
PMCID: PMC1745837  PMID: 10899249
10.  Bronchoalveolar lavage in ankylosing spondylitis. 
Annals of the Rheumatic Diseases  1990;49(5):325-326.
Bronchoalveolar lavage and bronchial biopsies were performed in 15 patients with ankylosing spondylitis (AS) and 17 control subjects. There was no difference in total cell count, number of lymphocytes, CD4+/CD8+ ratio, or beta 2 microglobulin concentrations in bronchoalveolar lavage fluid between these two groups. Bronchoalveolar lavage IgA concentrations were not increased, but bronchial IgA deposits were more common in AS. This study failed to show any subclinical alveolitis in AS.
PMCID: PMC1004080  PMID: 2140499
11.  Airway Lipoxin A4 Generation and Lipoxin A4 Receptor Expression Are Decreased in Severe Asthma 
Rationale: Airway inflammation is common in severe asthma despite antiinflammatory therapy with corticosteroids. Lipoxin A4 (LXA4) is an arachidonic acid–derived mediator that serves as an agonist for resolution of inflammation.
Objectives: Airway levels of LXA4, as well as the expression of lipoxin biosynthetic genes and receptors, in severe asthma.
Methods: Samples of bronchoalveolar lavage fluid were obtained from subjects with asthma and levels of LXA4 and related eicosanoids were measured. Expression of lipoxin biosynthetic genes was determined in whole blood, bronchoalveolar lavage cells, and endobronchial biopsies by quantitative polymerase chain reaction, and leukocyte LXA4 receptors were monitored by flow cytometry.
Measurements and Main Results: Individuals with severe asthma had significantly less LXA4 in bronchoalveolar lavage fluids (11.2 ± 2.1 pg/ml) than did subjects with nonsevere asthma (150.1 ± 38.5 pg/ml; P < 0.05). In contrast, levels of cysteinyl leukotrienes were increased in both asthma cohorts compared with healthy individuals. In severe asthma, 15-lipoxygenase-1 mean expression was decreased fivefold in bronchoalveolar lavage cells. In contrast, 15-lipoxgenase-1 was increased threefold in endobronchial biopsies, but expression of both 5-lipoxygenase and 15-lipoxygenase-2 in these samples was decreased. Cyclooxygenase-2 expression was decreased in all anatomic compartments sampled in severe asthma. Moreover, LXA4 receptor gene and protein expression were significantly decreased in severe asthma peripheral blood granulocytes.
Conclusions: Mechanisms underlying pathological airway responses in severe asthma include lipoxin underproduction with decreased expression of lipoxin biosynthetic enzymes and receptors. Together, these results indicate that severe asthma is characterized, in part, by defective lipoxin counterregulatory signaling circuits.
doi:10.1164/rccm.200801-061OC
PMCID: PMC2542432  PMID: 18583575
severe asthma; lipoxins; eicosanoids
12.  Desensitisation of neutrophil responses by systemic interleukin 8 in cystic fibrosis. 
Thorax  1994;49(9):867-871.
BACKGROUND--Inflammation associated with neutrophil infiltration is a commonly observed feature of children with cystic fibrosis. Production of the major neutrophil chemotactic cytokine interleukin 8 (IL-8) is potentially of great importance in the pathology of cystic fibrosis. Concentrations of IL-8 in both sputum and bronchoalveolar lavage fluid have been found to be higher in children with cystic fibrosis than in controls. The IL-8 induced chemotactic response and numbers of IL-8 receptors on peripheral neutrophils obtained from children with cystic fibrosis have been compared with a control group of children. METHODS--Cells were isolated from 18 patients with cystic fibrosis (aged 4-20 years) and 13 controls (aged 5-12 years) by dextran centrifugation followed by separation on Lymphoprep. Chemotaxis was assayed using multiwell microchemotaxis chambers and 5 microns polycarbonate filters. Filters were fixed and stained with Haema-Gurr for counting. Results were expressed as numbers of neutrophils per high power field (HPF). RESULTS--At the optimum concentration (1 x 10(-8) mol/l) the number of cells migrating were similar for controls (150 (12)/HPF) and for the cystic fibrosis group (140 (14)/HPF)). At lower concentrations the numbers of neutrophils migrating were lower for the cystic fibrosis group. Scatchard analysis of 125I-labelled IL-8 binding revealed lower numbers of receptors on neutrophils from patients with cystic fibrosis (22,000 per cell) than from controls (75,000 per cell). CONCLUSIONS--Reduced responsiveness to IL-8 of neutrophils from patients with cystic fibrosis is associated with receptor desensitisation as a result of exposure to high systemic levels of IL-8.
PMCID: PMC475175  PMID: 7940424
13.  Immunological features of lung lavage cells from patients with primary biliary cirrhosis may reflect those seen in pulmonary sarcoidosis. 
Gut  1990;31(2):208-212.
To investigate the basis of subclinical alveolitis in patients with primary biliary cirrhosis, 10 primary biliary cirrhosis patients were studied by bronchoalveolar lavage. Both bronchoalveolar lavage lymphoid and non-lymphoid cell populations were analysed using immunocytological methods to determine their proportions and phenotypic features in an attempt to gain information as to possible immune mechanisms active in the lung of these patients. Six of the 10 patients in our study showed evidence of an alveolitis (raised lymphocyte count: 27.6 (4.3)% of total count) on lavage. The results were compared with control groups of normal volunteers and patients with active pulmonary sarcoidosis. The six primary biliary cirrhosis patients with lymphocytosis had a raised CD4/CD8 T-cell ratio (4.13:1), similar to the sarcoid patients (5.60:1). A proportion of these T-lymphocytes expressed markers of activation (HLA-DR+ 7.5 (2.1)%); CD25 + 2.3 (0.9)%; CD7 + 5.8 (1.5)%. This increased T-cell activation was also seen in the sarcoid groups (HLA-DR+ 10.0 (1.9)%; CD25 + 3.0 (1.1)%; CD7 + 5.0 (0.2)%). This was not seen in the primary biliary cirrhosis patients without lymphocytosis and the normal volunteers. Within the non-lymphoid cell population, an increase in dendritic (RFD1+) cells was seen in primary biliary cirrhosis patients with lymphocytosis (31.2 (1.9)%) and sarcoid patients (46.3 (5.1)%) in contrast with the normal and primary biliary cirrhosis group without lymphocytosis. The primary biliary cirrhosis patients without lymphocytes had a relatively greater proportion of mature phagocytes (RFD7+). We postulate that these observations suggest the emergence in the lung of a granuloma producing mechanism similar to that occurring in the liver. By comparison, the alveolitis found in primary biliary cirrhosis is consistent with that observed in interstitial granulomatous lung disorders such as sarcoidosis.
PMCID: PMC1378382  PMID: 2311980
14.  Airway inflammatory markers in individuals with cystic fibrosis and non-cystic fibrosis bronchiectasis 
Bronchiectasis is an airway disease characterized by thickening of the bronchial wall, chronic inflammation , and destruction of affected bronchi. Underlying etiologies include severe pulmonary infection and cystic fibrosis (CF); however, in a substantial number of patients with non-CF-related bronchiectasis (NCFB), no cause is found. The increasing armamentarium of therapies now available to combat disease in CF is in stark contrast to the limited tools employed in NCFB. Our study aimed to evaluate similarities and differences in airway inflammatory markers in patients with NCFB and CF, and to suggest potential common treatment options. The results of this study show that NCFB bronchoalveolar lavage fluid samples possessed significantly increased NE activity and elevated levels of matrix metalloproteinases 2 (MMP-2) and MMP-9 compared to healthy controls (P < 0.01); however, the levels detected were lower than in CF (P < 0.01). Interleukin-8 (IL-8) concentrations were significantly elevated in NCFB and CF compared to controls (P < 0.05), but in contrast, negligible levels of IL-18 were detected in both NCFB and CF. Analogous concentrations of IL-10 and IL-4 measured in NCFB and CF were statistically elevated above the healthy control values (P < 0.05 and P < 0.01, respectively). These results indicate high levels of important proinflammatory markers in both NCFB and CF and support the use of appropriate anti-inflammatory therapies already employed in the treatment of CF bronchiectasis in NCFB.
doi:10.2147/JIR.S40081
PMCID: PMC3576001  PMID: 23426081
bronchiectasis; cystic fibrosis; proteases; inflammation
15.  Effect of natural allergen exposure during the grass pollen season on airways inflammatory cells and asthma symptoms. 
Thorax  1996;51(6):575-581.
BACKGROUND: Bronchial challenge with allergen causes a specific form of airways inflammation consisting of an influx of neutrophils, eosinophils, and T cells. Because the relevance of the challenge model to clinical asthma is uncertain, the cellular changes that occur in the lungs of asthmatic subjects during natural seasonal allergen exposure were investigated. METHODS: Seventeen grass pollen sensitive asthmatic subjects with previously reported seasonal exacerbations of asthma kept records of symptoms and underwent fibreoptic bronchoscopy with bronchoalveolar lavage (BAL) and endobronchial biopsy before and during the peak of the grass pollen season. The BAL cells were analysed for differential cell counts and by flow cytometry for T cell subsets and surface activation markers. The biopsy samples were processed into glycol methacrylate resin and immunohistochemical analysis was performed for mast cells, activated eosinophils, T cells and interleukin 4 (IL-4), a cytokine with a pivotal role in allergen-induced inflammation. RESULTS: In the pollen season there was an increase in T lymphocyte activation in the BAL fluid as identified by increased expression of interleukin 2 receptor (IL-2R). In the submucosa these changes were paralleled by an increase in CD4+ T cells. By contrast, the numbers of metachromatic cells in BAL fluid staining with toluidine blue were reduced, possibly because of degranulation following allergen stimulation. In keeping with mast cell activation, the number of mucosal mast cells staining for secreted IL-4 increased during the season. In comparison with the period shortly before the onset of the season, all but two subjects experienced an asthma exacerbation which followed the rise in pollen counts but, compared with the period preceding the first bronchoscopic examination, asthma symptoms were not increased during the pollen season. CONCLUSIONS: The data suggest that natural allergen exposure, leading to a clinical exacerbation of asthma, may induce an inflammatory response involving T cells, mast cells and eosinophils. The relationship between allergen exposure, cellular infiltration and activation, and clinical symptoms appears to be complex, with factors other than allergen also contributing to asthmatic activity.
PMCID: PMC1090485  PMID: 8693436
16.  Phenotype of airway epithelial cells suggests epithelial to mesenchymal cell transition in clinically stable lung transplant recipients 
Thorax  2005;60(10):865-871.
Background: Obliterative bronchiolitis in chronic rejection of lung allografts is characterised by airway epithelial damage and fibrosis. The process whereby normal epithelium is lost and replaced by fibroblastic scar tissue is poorly understood, but recent findings suggest that epithelial cells can become fibroblasts through epithelial-mesenchymal transition (EMT). It is hypothesised that EMT occurs in lung allografts and plays a potential role in airway remodelling.
Methods: Sixteen stable lung transplant recipients underwent bronchoscopy with bronchoalveolar lavage (BAL), endobronchial biopsies, and bronchial brushings. Biopsy sections were stained for the fibroblast marker S100A4. Brushings were cultured on collagen, stained with anti-S100A4, and examined for further EMT markers including matrix metalloproteinase (MMP) zymographic activity and epithelial invasion through collagen coated filters.
Results: A median 15% (0–48%) of the biopsy epithelium stained for S100A4 in stable lung transplant recipients and MMP-7 co-localisation was observed. In non-stimulated epithelial cultures from lung allografts, S100A4 staining was identified with MMP-2 and MMP-9 production and zymographic activity. MMP total protein and activity was increased following stimulation with transforming growth factor (TGF)-ß1. Non-stimulated transplant epithelial cells were invasive and penetration of collagen coated filters increased following TGF-ß1 stimulation.
Conclusions: This study provides evidence of EMT markers in lung allografts of patients without loss of lung function. The EMT process may represent a final common pathway following injury in more common diseases characterised by airway remodelling.
doi:10.1136/thx.2005.043026
PMCID: PMC1747194  PMID: 15972366
17.  Latent pulmonary involvement in Crohn's disease: biological, functional, bronchoalveolar lavage and scintigraphic studies. 
Gut  1986;27(8):919-925.
We have investigated the following pulmonary related parameters in 22 patients with Crohn's disease who were free of clinical pulmonary symptoms and had normal chest roentgenograms and in 25 controls: serum angiotensin converting enzyme, pulmonary function tests, bronchoalveolar lavage (lymphocyte count and subpopulations, macrophage viability and superoxide anion release by macrophages) and pulmonary scannings. Serum angiotensin converting enzyme was lower in Crohn's disease (14.1 +/- 5.1) than in controls (25.2 +/- 4.7) (p less than 0.001). Twelve of 22 Crohn's disease (54%) had a bronchoalveolar lavage lymphocytosis (greater than 18% alveolar lymphocytes). Bronchoalveolar lavage lymphocytes subpopulations were quite variable. Twelve of 17 Crohn's disease (71%) had an increase spontaneous and/or stimulated superoxide anion production by alveolar macrophages. Six of 12 Crohn's disease (50%) had an increase physiologic dead space in the upper part of their lung against one of 11 controls (9%). These data suggest that most patients with Crohn's disease have a latent pulmonary involvement.
PMCID: PMC1433358  PMID: 3015749
18.  Analysis of bronchoalveolar lavage fluid in patients with chronic hepatitis C before and after treatment with interferon alpha 
Thorax  1997;52(1):33-37.
BACKGROUND: Previous studies have shown that patients with idiopathic pulmonary fibrosis (IPF) were more likely to be seropositive for hepatitis C virus (HCV) than normal controls, and that patients with chronic hepatitis C treated with interferon alpha (IFN-alpha) sometimes developed pulmonary fibrosis. The possibility that HCV infection and/or treatment with IFN-alpha are involved in the pathogenesis of pulmonary fibrosis or alveolitis was investigated. METHODS: A prospective non- randomised study was performed in 13 healthy controls and in patients with chronic hepatitis C before (n = 13) and after (n = 10) treatment with IFN-alpha. Bronchoalveolar lavage (BAL) fluid cell counts, ratios and T cell subsets, and the concentrations of interleukin (IL)-1 beta, tumour necrosis factor(TNF)-alpha, and hepatocyte growth factor (HGF) were measured. RESULTS: Lymphocyte counts in the BAL fluid were significantly increased in both groups of patients (median (range) values: before treatment, 36.8 (1.5-226.0); after treatment, 16.2 (4.5- 97.6)) compared with the normal controls (3.3 (0.5-32.3)). In the pretreatment group the activated T cell (HLA-Dr positive) count was also increased (51 (40-74)) compared with that in the normal controls (27 (4-52)), but after treatment it was decreased (40 (0-76)) compared with the pretreatment count. Administration of IFN-alpha did not affect these parameters. IL-1 beta, TNF-alpha, and HGF were not detected. CONCLUSIONS: These findings suggest that HCV infection is associated with increased counts of lymphocytes and neutrophils in BAL fluid and that treatment with IFN-alpha appears to alter lymphocyte surface markers. 



PMCID: PMC1758417  PMID: 9039237
19.  Plasminogen Is an Important Regulator in the Pathogenesis of a Murine Model of Asthma 
Rationale: Asthma is a syndrome whose common pathogenic expression is inflammation of the airways. Plasminogen plays an important role in cell migration and is also implicated in tissue remodeling, but its role in asthma has not been defined.
Objectives: To test whether plasminogen is a critical component in the development of asthma.
Methods: We used a mouse model of ovalbumin-induced pulmonary inflammation in Plg+/+, Plg+/−, and Plg−/− mice.
Measurements and Main Results: The host responses measured included lung morphometry, and inflammatory mediators and cell counts were assessed in bronchoalveolar lavage fluid. Bronchoalveolar lavage demonstrated a marked increase in eosinophils and lymphocytes in ovalbumin-treated Plg+/+ mice, which were reduced to phosphate-buffered saline–treated control levels in Plg+/− or Plg−/− mice. Lung histology revealed peribronchial and perivascular leukocytosis, mucus production, and increased collagen deposition in ovalbumin-treated Plg+/+ but not in Plg+/− or Plg−/− mice. IL-5, tumor necrosis factor-α, and gelatinases, known mediators of asthma, were detected in bronchoalveolar lavage fluid of ovalbumin-treated Plg+/+ mice, yet were reduced in Plg−/− mice. Administration of the plasminogen inhibitor, tranexamic acid, reduced eosinophil and lymphocyte numbers, mucus production, and collagen deposition in the lungs of ovalbumin-treated Plg+/+ mice.
Conclusions: The decreased inflammation in the lungs of Plg−/− mice and its blockade with a plasminogen inhibitor indicate that plasminogen plays an important role in orchestrating the asthmatic response and suggests that plasminogen may be a therapeutic target for the treatment of asthma.
doi:10.1164/rccm.200609-1345OC
PMCID: PMC1994216  PMID: 17541016
lung; knockout mice; pulmonary inflammation; fibrinolysis
20.  Chronic pulmonary sarcoidosis: relationship between lung lavage cell counts, chest radiograph, and results of standard lung function tests. 
Thorax  1985;40(7):501-507.
Thirty three consecutive untreated patients with pulmonary sarcoidosis, confirmed histologically or by Kveim test, were investigated to correlate cell counts in bronchoalveolar lavage fluid with clinical features, the chest radiograph, and results of lung function tests. A persistently abnormal radiograph had been observed for one year or more in 26 (79%) and for two years or more in 20 (61%), but only 24% had dyspnoea. Twenty (61%) of 33 patients showed an increased percentage of lymphocytes in bronchoalveolar lavage fluid, although only eight (24%) exceeded 28%. A moderate increase of neutrophils, up to 12%, was found in 14 (42%). Lymphocyte percentage counts were higher in the group of patients without evidence of radiographic contraction suggesting fibrosis, and this contrasted with higher percentage neutrophil counts in those with contraction. There was also a correlation between the percentages of neutrophils and increasing radiographic profusion scores (p less than 0.001), suggesting that neutrophils may reflect the severity of the parenchymal legions as well as fibrotic distortion, and an inverse correlation with vital capacity (p less than 0.001) and transfer factor (TLCO) (p less than 0.1 greater than 0.05). No significant correlation was found between the lymphocyte counts and radiographic profusion scores, vital capacity or TLCO; but it was noted that all eight patients with high lymphocyte counts (greater than 28%) had radiographic profusion scores less than 12. This study shows that, especially in sarcoidosis with more extensive radiographic shadows of long duration, bronchoalveolar lavage neutrophils may be important as well as lymphocytes in clinical assessment of "activity" of disease. These observations are important because they throw doubt on whether the lavage lymphocyte count alone can be used as an indicator of the need to start corticosteroid treatment.
PMCID: PMC460121  PMID: 4035616
21.  Airway neutrophilia in stable and bronchiolitis obliterans syndrome patients following lung transplantation 
Thorax  2000;55(1):53-59.
BACKGROUND—The bronchiolitis obliterans syndrome (BOS) remains the major constraint on the long term success of lung transplantation. Neutrophils have been associated with fibrosing lung conditions and have been noted to be increased in the bronchoalveolar lavage (BAL) fluid of patients with BOS.
METHODS—This study was undertaken to examine neutrophil accumulation in the BAL fluid, airway wall and lung parenchyma, as well as levels of interleukin (IL)-8 in the BAL fluid, in normal controls and lung transplant recipients with and without BOS. Bronchoscopic examination included endobronchial biopsy (EBB), BAL fluid, and transbronchial biopsy (TBB) sampling. Tissue neutrophils were identified by neutrophil elastase staining on 3 µm paraffin biopsy sections and quantified by computerised image analyser. IL-8 levels were measured in unconcentrated BAL fluid by ELISA.
RESULTS—Compared with controls, airway wall neutrophilia was increased in both stable lung transplant recipients and those with BOS (p<0.05). BAL neutrophils and IL-8 levels were also increased in both groups of transplant recipients compared with controls (p<0.01), the levels being significantly higher in the BOS group (p<0.01). Neutrophil numbers in the lung parenchyma were not significantly different between the two groups of lung transplant recipients.
CONCLUSION—Increased levels of neutrophils are present in the airway wall and BAL fluid of lung transplant recipients with and without BOS. BAL fluid levels of IL-8 are also increased, raising the possibility that neutrophils and/or IL-8 may play a part in the pathogenesis of BOS following lung transplantation.


doi:10.1136/thorax.55.1.53
PMCID: PMC1745588  PMID: 10607802
22.  Pseudomonas aeruginosa exoenzyme S induces proliferation of human T lymphocytes. 
Infection and Immunity  1995;63(5):1800-1805.
Pseudomonas aeruginosa is a gram-negative bacterium that is responsible for devastating acute and chronic infections, which include bronchiectasis in cystic fibrosis, nosocomial pneumonia, and infection of burn wounds. Previous studies have demonstrated that these patients have impaired host responses, including cell-mediated immune responses, which are important in anti-Pseudomonas host defense. The P. aeruginosa exoproduct, exoenzyme S, has a number of characteristics which suggest that it might be important in cell-mediated immunity. To determine whether exoenzyme S activates lymphocytes to proliferate, peripheral blood mononuclear cells (PBMC) from normal volunteers were stimulated with purified exoenzyme S, and the lymphocyte response was assessed by measuring [3H]thymidine uptake and by counting the number of cells after various times in culture. Ninety-five percent of healthy adult donors had a lymphocyte response to exoenzyme S. The optimal lymphocyte response occurred on day 7, with 4 x 10(5) PBMC per microtiter well when cells were stimulated with 10 micrograms exoenzyme S per ml. [3H]thymidine uptake correlated with an increase in the number of mononuclear cells, indicating that proliferation occurred. In unseparated PBMC, T cells, and to a lesser extent B cells, proliferated. Purified T cells proliferated, while purified B cells proliferated only after the addition of irradiated T cells. Thus, T lymphocytes are necessary and sufficient for the proliferative response to exoenzyme S. We speculate that exoenzyme S from P. aeruginosa is important in T-lymphocyte-mediated host defense to P. aeruginosa. In strategies to enhance impaired cell-mediated immunity, exoenzyme S should be considered as a potential stimulant.
PMCID: PMC173227  PMID: 7537248
23.  Protective Role of Andrographolide in Bleomycin-Induced Pulmonary Fibrosis in Mice 
Idiopathic pulmonary fibrosis (IPF) is a chronic devastating disease with poor prognosis. Multiple pathological processes, including inflammation, epithelial mesenchymal transition (EMT), apoptosis, and oxidative stress, are involved in the pathogenesis of IPF. Recent findings suggested that nuclear factor-κB (NF-κB) is constitutively activated in IPF and acts as a central regulator in the pathogenesis of IPF. The aim of our study was to reveal the value of andrographolide on bleomycin-induced inflammation and fibrosis in mice. The indicated dosages of andrographolide were administered in mice with bleomycin-induced pulmonary fibrosis. On day 21, cell counts of total cells, macrophages, neutrophils and lymphocytes, alone with TNF-α in bronchoalveolar lavage fluid (BALF) were measured. HE staining and Masson’s trichrome (MT) staining were used to observe the histological alterations of lungs. The Ashcroft score and hydroxyproline content of lungs were also measured. TGF-β1 and α-SMA mRNA and protein were analyzed. Activation of NF-κB was determined by western blotting and electrophoretic mobility shift assay (EMSA). On day 21 after bleomycin stimulation, andrographolide dose-dependently inhibited the inflammatory cells and TNF-α in BALF. Meanwhile, our data demonstrated that the Ashcroft score and hydroxyproline content of the bleomycin-stimulated lung were reduced by andrographolide administration. Furthermore, andrographloide suppressed TGF-β1 and α-SMA mRNA and protein expression in bleomycin-induced pulmonary fibrosis. Meanwhile, andrographolide significantly dose-dependently inhibited the ratio of phospho-NF-κB p65/total NF-κB p65 and NF-κB p65 DNA binding activities. Our findings indicate that andrographolide compromised bleomycin-induced pulmonary inflammation and fibrosis possibly through inactivation of NF-κB. Andrographolide holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.
doi:10.3390/ijms141223581
PMCID: PMC3876064  PMID: 24300094
andrographolide; bleomycin (BLM); pulmonary fibrosis; transforming growth factor-β1 (TGF-β1); alpha-smooth muscle actin (α-SMA); nuclear factor-κB (NF-κB)
24.  Bronchoalveolar lavage fluid findings in patients with chronic hepatitis C virus infection. 
Thorax  1996;51(3):312-314.
BACKGROUND: Hepatitis C virus (HCV) infection has recently been incriminated as an aetiological agent in idiopathic pulmonary fibrosis. This study was performed to determine the cellularity and lymphocyte phenotypes of bronchoalveolar lavage (BAL) fluid in patients with chronic hepatitis C. METHODS: BAL fluid and lavage lymphocyte subsets from 13 patients (10 men) with active chronic hepatitis C, diagnosed by sustained elevated serum glutamic pyruvic transaminase and typical histological findings in the liver, were analysed. Lavage findings in these patients were compared with those from 13 healthy volunteers (eight men) as controls. RESULTS: There was no difference in total cell counts in lavage fluid between the two groups. Lavage lymphocyte and eosinophil numbers were increased in patients with chronic hepatitis C. Surface marker analysis of the lymphocyte populations showed increases in CD2, CD3, CD4, and HLA-DR. CD4/CD8 ratios were not different. CONCLUSIONS: The numbers of lymphocytes and eosinophils in BAL fluid are increased in patients with chronic hepatitis C. These findings suggest that HCV infection may trigger alveolitis.
PMCID: PMC1090646  PMID: 8779138
25.  Bronchoalveolar lavage in pulmonary fibrosis: comparison of cells obtained with lung biopsy and clinical features 
Thorax  1980;35(1):9-18.
Bronchoalveolar lavage, open lung biopsy, and cell extraction from the biopsy material have been studied in 21 symptomatic patients with progressive pulmonary fibrosis (18 with cryptogenic fibrosing alveolitis, fulfilling also the criteria for “usual interstitial pneumonia” (UIP), and three with rapidly progressive disease probably related to asbestos exposure). The total and differential cell counts between the three different samples have been compared as well as the influence on them of smoking and their correlation with steroid responsiveness and later progress. There was no correlation between semiquantitative scores of cell types observed within alveolar spaces and in alveolar walls and the differential or total cell counts obtained from extraction or lung lavage samples. There was, however, some correlation between differential counts obtained from lung lavage and extractions (neutrophils p<0·02, eosinophils p<0·07, lymphocytes p<0·08) suggesting that lung lavage reflects the cellularity of the peripheral parts of the lung in patients without overt bronchial disease. Steroid responsiveness related to the percentage of lymphocytes found in extraction samples (p<0·01) and was associated with a complementary fall in the percentage of macrophages (p<0·02). There was no relationship between steroid response and the numbers of neutrophils or eosinophils in extracted samples. There was a trend towards increased numbers of lymphocytes in the lung wash in those patients responding to steroids. Those cases showing more rapid progression before starting treatment tended to have higher percentages of lymphocytes, neutrophils, or eosinophils in the lung lavage than more slowly deteriorating cases (p<0·01). Follow-up studies showed that three cases having predominant lymphocytes in the lung lavage continued to do well while nine cases with predominant neutrophils or eosinophils or both showed a less satisfactory response to steroids and often deteriorated. Differential cell counts from biopsy extractions and lung lavage may give information additional to conventional light microscopy on the likelihood of steroid responsiveness as well as providing some measure of the activity of disease.
PMCID: PMC471213  PMID: 7361291

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