Relationship between lipid peroxidase and superoxide dismutase in bronchoalveolar lavage fluid and pulmonary qi deficiency syndrome

Investigating the levels of lipid peroxidase (LPO) and superoxide dismutase (SOD) in serum, bronchoalveolar lavage fluid (BALF) and alveolar macrophage (AM) were determined in 40 chronic bronchitis patients with Pulmonary Qi Deficiency (PQD) Syndrome and 36 normal subjects. Results showed: (1) No significant differences were found between PQD syndrome and normal subjects on serum SOD or LPO levels. (2) Patients with PQD Syndrome. LPO level in BALF was significantly higher, and SOD significantly lower, when compared with normal subjects. (3) Through correlation analysis, it was found in BALF that SOD level was markedly correlated with AM, while LPO was significantly correlated with neutrophil. In short, SOD and LPO in BALF play an important role in the development of the PQD Syndrome, and are good indications in evaluating the PQD Syndrome.     

Increased gelatinolytic activity in bronchoalveolar lavage fluid in stable lung transplant recipients

Proteolytic enzymes have been proposed to play a role in the pathogenesis of various inflammatory pulmonary diseases accompanied by parenchymal remodeling. To assess the role of inflammatory cells and proteolytic enzymes in the development of chronic allograft rejection after lung transplantation, bronchoalveolar lavage fluid (BALF) samples from clinically stable lung transplant (LT) recipients (i.e., without evidence of active infection or rejection), heart transplant (HT) recipients, and healthy volunteers (NL) were analyzed for total white blood cell (WBC) count and differential cell count, along with gelatinolytic/type IV collagenolytic activity. The LT group displayed a significantly increased total WBC count, neutrophil count, and percent neutrophils compared with the NL group, confirming the presence of inflammation. Furthermore, gelatin zymography revealed a significant increase in activity of the 72 and 92 kD gelatinases in the LT group compared with the NL group. A positive correlation existed between neutrophil counts and the increase in proteolytic activity. Immunosuppressive therapy did not account for the findings, since no significant difference in cell counts or proteolytic activity existed between the NL and HT control groups. These findings, together with those of others that relate chronic lung allograft dysfunction to an increase in BALF neutrophils and collagen matrix remodeling, collectively indicate that up-regulated proteolytic activity may have a role in chronic rejection after lung transplantation.     

Peptide amidating activity in human bronchoalveolar lavage fluid

Monitoring respiratory epithelial biology may reveal individuals with incipient lung cancer. The expression of neuroendocrine (NE) markers in pulmonary epithelium is thought to be central to lung development, repair of injury and may contribute to carcinogenesis. In this study, we evaluate several candidate NE markers to determine the feasibility of prospective analysis of clinical specimens. The potential NE markers include the enzyme L-DOPA decarboxylase (DDC), the neuropeptide gastrin releasing peptide (GRP), and peptidyl-glycine alpha-amidating monooxygenase (PAM), the bifunctional enzyme responsible for the final bioactivation step of many neuropeptides. A comparison of PAM activity and DDC levels in 30 lung cancer cell lines indicated that peptide amidating activity may be an indicator of NE status. Bronchoalveolar lavage (BAL) fluid from subjects at risk of developing second primary lung cancer and from volunteers was obtained. The activity of the first PAM enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), ranged from not detectable to 507 pmol/h/mg protein in 57 specimens. The second PAM enzyme, peptidylamidoglycolate lyase (PAL), ranged from not detectable to 414 pmol/h/mg protein in 56 specimens. Using cluster analysis by the average linkage method, a group of enzyme values with PHM greater than 230 pmol/h/mg protein was determined. Long-term follow-up of these patients for new second primary lung cancers may help to determine the potential predictive value of PAM detected in the BAL fluid.     

Segmental antigen challenge increases fibronectin in bronchoalveolar lavage fluid

Fibronectin may contribute to asthma pathogenesis by recruitment and activation of inflammatory cells, and by promotion of subepithelial fibrosis. Fibronectin is produced by several types of airway cells, including epithelial cells, fibroblasts, and alveolar macrophages. To test the hypothesis that antigen-induced airway inflammation is associated with increased local generation of fibronectin, segmental bronchoprovocation (SBP) with antigen and saline was performed in 17 atopic patients. Bronchoalveolar lavage (BAL) was performed at 5 min and 48 h after segmental challenge with saline or antigen. Fibronectin concentrations in BAL fluid, measured by enzyme-linked immunosorbent assay (ELISA), increased more than 5-fold 48 h after antigen challenge (65 [47 to 110] versus 407 [240 to 697] ng/ml, median and 25 to 75% interquartiles, p < 0.05). Fibronectin concentrations 48 h after antigen challenge correlated with histamine concentrations 5 min after antigen challenge and numbers of eosinophils, neutrophils, macrophages, and total cells in BAL fluid 48 h after antigen challenge. BAL was more enriched in fibronectin 48 h after challenge than would be predicted solely from increased permeability of plasma proteins. Western blot analysis showed that fibronectin in BAL fluid was largely intact and contained the extra domain-A (ED-A) splice variant of cellular fibronectin, indicative of local production. We conclude that antigen challenge in atopic subjects causes increased production of fibronectin by airway cells and speculate that this response may contribute to airway remodeling in allergic inflammation.     

Immunoglobulin levels in bronchoalveolar lavage fluid from pigeon breeders

Analysis of serum and BAL fluid immunoglobulin levels in individuals with PBD and in asymptomatic but similarly exposed pigeon breeders was carried out by immunofluorometric assays. The results indicate that the group with PBD have significantly higher levels of IgG and IgA in their BAL fluids but that IgM levels were not significantly different in the two groups. These differences were not reflected in the serum immunoglobulin levels of the two groups. The elevated BAL fluid IgG levels in individuals with PBD is associated with an increase in IgG4 subclass levels as determined PHA inhibition. These studies suggest a role for this subclass in the pathogenesis of the disease.     

Soluble complement receptor type 1 (CD35) in bronchoalveolar lavage of inflammatory lung diseases

The presence of epidermal-growth-factor receptors (EGFR) and of its ligands (TGFalpha and amphiregulin) in breast-cancer tissues suggests that they play a paracrine/autocrine role in tumor growth or progression. This hypothesis was tested on 3 cell lines, S2T2, NS2T2A and NS2T2A1. These epithelial cells are derived from a normal human breast-epithelial-cell culture transformed by SV40-T Ag, are of the same clonal origin, have respectively increasing levels of EGFR, TGFalpha, amphiregulin and of thymidine-kinase activity associated with increasing tumorigenic potential in nude mice (tumor intake and tumor volume). The monoclonal antibody MAb 425, which blocks ligands interaction with EGFR, reduced by more than 90% anchorage-independent growth of the most tumorigenic cells, NS2T2A1. Another anti-EGFR MAb, 528, reduced to 25% of controls the mean tumor mass after NS2T2A1 grafting in mice. Anti-sense RNA expression of EGFR in these cells confirmed the importance of this receptor in tumor progression, since it reduced significantly the tumor volume and tumor weight of NS2T2A1 cells to 16% of those in mock-transfected control cells.     

Non-fibrous inorganic particles in bronchoalveolar lavage fluid of pottery workers

AIM: To study the actual exposure of pottery workers to silica particles, as their risk of silicosis is potentially high because of the presence of inhalable crystalline silica particles in the workplace. METHODS: Nine pottery workers underwent bronchoalveolar lavage. The recovered fluid was analysed for cytological and mineralogical content by analytical transmission electron microscopy. The data were compared with those obtained from a control group composed of seven patients with sarcoidosis and six patients with haemoptysis. RESULTS: Cytological results showed a similar profile in exposed workers and controls, whereas in patients with sarcoidosis a lymphocytic alveolitis was found. Microanalysis of the particulate identified the presence of silicates, CRSs, and metals. Pottery workers had higher numbers of total particles and CRSs, and had a higher silicate/metal ratio. In five workers, the presence of zirconium silicate was also detected. Patients with sarcoidosis had the lowest number of particles, and an inverted silicate/metal ratio. CONCLUSION: Microanalysis by transmission electron microscope can provide useful information to assess occupational exposure to dusts.     

Soluble L-selectin concentration in bronchoalveolar lavage fluid obtained from infants who develop chronic lung disease of prematurity

AIMS: To explore the changes in neutrophil adhesion molecule expression and release into bronchoalveolar lavage fluid (BAL) obtained from infants who developed chronic lung disease (CLD). METHODS: BAL fluid was obtained from 37 infants: 18 (median gestation 26 weeks, birthweight 835 g) who developed CLD, 12 (29 weeks, 1345 g) with respiratory distress syndrome (RDS) and seven control infants (33 weeks, 2190 g). RESULTS: Soluble L-selectin (sL-selectin) in BAL fluid from the CLD and non-CLD groups was similar immediately after birth, but in infants who subsequently developed CLD, sL-selectin remained persistently increased (at day 7: CLD 42.6 vs RDS 6.0 ng/ml, p < 0.05; CLD vs controls 1.5 ng/ml; p < 0.05). CD11b/CD18 expression on neutrophils obtained by BAL increased with time to reach a maximum at 17 days of age in infants who developed CLD. CONCLUSIONS: These results suggest that leucocyte traffic persists in infants who develop CLD and may have an important part to play in the pathogenesis of CLD.     

Neurotrophins are increased in bronchoalveolar lavage fluid after segmental allergen provocation

The mechanisms linking inflammation and airway hyperresponsiveness in allergic bronchial asthma are still not completely defined. Since neurotrophic factors increase nerve excitability and neurotransmitter synthesis and are produced by immunocompetent cells, they are likely candidates as mediators of inflammation and hyperresponsiveness. We tested the hypothesis that neurotrophin concentrations will increase in the bronchoalveolar lavage (BAL) fluid from patients with asthma after segmental allergen provocation. For this purpose an individually standardized dose of allergen or saline was instilled into different segments during bronchoscopy in eight subjects with mild allergic bronchial asthma. Segments were then lavaged 10 min and 18 h after allergen challenge or saline instillation. There was a significant increase in the neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 in BAL fluids 18 h after allergen but not saline challenge. We conclude that neurotrophins are produced endobronchially following allergen provocation, suggesting a contribution to the pathogenesis of asthma.     

Pneumocystis carinii alters surfactant protein A concentrations in bronchoalveolar lavage fluid

To understand better the interaction between surfactant protein A (SP-A), human immunodeficiency virus (HIV) and Pneumocystis carinii pneumonia (PCP), we measured SP-A from bronchoalveolar lavage (BAL) fluid in immunosuppressed patients (HIV-positive [HIV+] and HIV noninfected [HIV-]) who were examined for possible pneumonia. Forty-five HIV+ patients, 16 with PCP and no other pathogen (HIV+/Pc) and 29 with no evidence of pulmonary pathogen (HIV+ controls), were compared with 6 HIV- patients with PCP (HIV-/Pc) and 11 control patients with no underlying disease (controls). Despite a similar inflammatory response in the HIV-infected patients whether they had PCP or not, we found increased BAL SP-A concentrations in HIV+/Pc patients as compared with HIV+ control patients (HIV+/Pc: median, 10.3 micrograms/ml; range, 2.8 to 24.3 micrograms/ml; HIV+ control: median, 1.9; range, 0.06 to 3.83 micrograms/ml; p < 0.05). The amount of SP-A in the HIV+ control group was significantly lower than healthy, uninfected volunteers, suggesting that HIV itself may lower SP-A levels. Six HIV+/Pc patients underwent BAL after 21 days of therapy and showed complete resolution of the P. carinii organism. There was a significant drop in the amount of SP-A at follow-up lavage (initial mean, 14.1 micrograms/ml; follow-up mean, 7.4 micrograms/ml; p < 0.02). We also found a significant correlation between the amount of P. carinii and the amount of SP-A in the BAL fluid (Spearman rank, 0.74; p < 0.01). We conclude that SP-A content is increased in HIV+ patients with PCP. The relationship between SP-A concentration and the abundance of P. carinii present in the BAL fluid may be related to SP-A binding to P. carinii or to alterations in surfactant protein homeostasis.     

Imbalance between Pneumocystis carinii cysts and trophozoites in bronchoalveolar lavage fluid from patients with pneumocystosis receiving prophylaxis

The effect of different culture conditions on thermostable lipase production by Bacillus sp. was studied in shake flasks. A maximum enzyme activity of 67-75 nkat/mL was observed in a medium consisting of 0.5% soybean flour and 0.1% stearyl glycerol esters or natural fats. A lipase activity of about 117 nkat/mL was established when the cultivation was carried out in a laboratory fermentor at 20% minimal dissolved oxygen level, the enzyme production being increased 1.5 fold compared to that in a flask culture.     

The clinical utility of asbestos body counts in bronchoalveolar lavage fluid

To assess the clinical utility of measuring the number of asbestos bodies (AB) present in bronchoalveolar lavage fluid (BALF), we counted the number of AB in BALF from 119 subjects using light microscopy. The results were analyzed according to occupational histories, radiological findings of asbestos-induced lung and pleural changes, and asbestos-related diseases. The 94 subjects in group 1 had a history of dust exposure, whereas group 2 subjects (n = 25) had no dust exposure. Group 1 was subdivided into subjects with obvious exposure to asbestos (group 1A, n = 61), and subjects with no known exposure to asbestos (group 1B, n = 33). The distribution of AB counts per ml of BALF (means +/- SEM) differed significantly between groups 1 and 2 (38.8 +/- 17.4 vs 0.06 +/- 0.04, p < 0.0001). The AB counts were significantly different between groups 1A and 1B (57.9 +/- 26.6 vs 3.4 +/- 1.2, p = 0.01). Subject, exposed to dust who had radiological evidence of pleural thickening had significantly higher AB counts than subjects in whom pleural thickening was absent (66.0 +/- 31.1 vs 5.1 +/- 4.2, p = 0.03). In group 1, the BALF was positive for AB in 7 of 14 patients with pulmonary fibrosis, 4 of 5 patients with lung cancer, all 6 patients with malignant mesothelioma, and all 4 patients with benign asbestos pleural effusion. We conclude that AB counts in BALF are useful for evaluating both the history of asbestos exposure in a population exposed to dust, as well as patients having asbestos-related diseases.     

Mast cell: it''s significance in bronchoalveolar lavage fluid cytologic diagnosis of bronchial asthma and interstitial lung disease]

Insect class I glutathione S-transferases (GSTs) were expressed from cDNA obtained from larvae of the Thai malaria vector. Anopheles dirus in a PCR RACE (rapid amplification of cDNA ends) reaction using a primer to the conserved N-terminal region of An. gambiae class I GSTs and a synthetic oligo d(T)-adaptor primer. Seven different plasmids, resulting from sub-cloning of an original single 0.7 Kb PCR band, were picked at random and sequenced. Four of these were clearly GSTs on the basis of putative amino acid sequence conservation. All the sequences had a conserved N-terminal region, but were highly divergent at the C-terminus. The variability in the PCR products suggests that there is a high level of GST class I isoenzyme variability in larval An. dirus. One of the subclones from the PCR reaction contained a full coding region of the cDNA for GST. This had a putative amino acid sequence which was 76 and 91% identity to the An. gambiae GST class I, agGST 1-5 and agGST 1-6 respectively, but only 48% identity to agGST 1-2. The catalytically active enzyme, expressed in Escherichia coli, was strongly immuno-cross reactive with antisera raised against the two An. gambiae class I GSTs. The expressed enzyme was purified to homogeneity from an E. coli cell lysate by S-hexylglutathione agarose affinity chromatography. The enzyme had a high specific activity with CDNB, and also used DCNB and ethacrynic acid as substrates. In addition, it had peroxidase and DDTase activity and its activity with CDNB, was strongly inhibited by a range of organophosphorus and pyrethroid insecticides. This is consistent with the predicted role of this GST class in insecticide resistance.     

Neutrophil chemokines in bronchoalveolar lavage fluid and leukocyte-conditioned medium from nonsmokers and smokers

Polymorphonuclear neutrophils (PMN) have been implicated in the pathogenesis of emphysema. The chemokines interleukin-8(IL-8), growth-related oncogene (GRO-alpha) and extractable nuclear antigen (ENA)-78 may be involved in the increased numbers of PMN in smokers' airspaces. The levels of these cytokines in bronchoalveolar lavage fluid (BALF) and bronchoalveolar lavage leukocyte conditioned medium (LCM), along with BALF PMN numbers in 12 smokers who abstained for 12 h (chronic smoking) or continued to smoke until I h before study (acute smoking) and seven nonsmokers were compared. Neutrophils in BALF increased in acute (1.96+/-0.53%, 0.99+/-0.32x10(6) cells) compared with chronic smokers (0.59+/-0.25%, 0.61+/-0.24x10(6) cells, p<0.05 nonsmokers) and nonsmokers (0.79+/-0.29%, 0.05+/-0.01x 10(6) cells, p<0.05). There were no differences in IL-8 or GRO-alpha in BALF between smokers and nonsmokers. ENA-78 levels were lower in smokers (p=0.006). There was no difference in IL-8, GRO-alpha or ENA-78 in LCM from unstimulated cells in smokers versus nonsmokers. After stimulation with lipopolysaccharide (LPS) 10 ng mL(-1), IL-8 release in acute smokers (p=0.04) and GRO-alpha release in smokers (p=0.009) were significantly higher than in nonsmokers. Following stimulation with LPS 100 ng.mL(-1), GRO-alpha release was higher in smokers (p=0.03) and increased further in acute smokers (p=0.02 versus nonsmokers, p=0.04 versus chronic smokers) and ENA-78 release increased in smokers (p=0.02 versus non-smokers). In conclusion, influx of polymorphonuclear neutrophils into smokers' airspaces is an acute phenomenon and neutrophil chemokine release from mixed bronchoalveolar lavage leukocytes is influenced by cigarette smoking and endotoxins.     

Detection of anionic antimicrobial peptides in ovine bronchoalveolar lavage fluid and respiratory epithelium

Three small antimicrobial anionic peptides (AP) were originally isolated from an ovine pulmonary surfactant. However, their presence in bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract is unknown. In this study, we made affinity-purified rabbit polyclonal and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody specificity was assessed by a competitive enzyme-linked immunosorbent assay (ELISA), and the exact epitope binding sites were determined with analog peptides synthesized on derivatized cellulose. These antibodies were used to detect AP in BAL fluid by ELISA and in respiratory tissues by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 +/- 0.33 mM AP (mean +/- standard deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 micrograms/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and 25.7 kDa). Only a single band was seen in solubilized liver and small-intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, or kidney or spleen homogenates. In pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial cells, and in an occasional alveolar macrophage. As a first step in identifying a candidate AP precursor gene(s), degenerate oligonucleotides representing all possible coding combinations for H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe Southern blots of sheep genomic DNA. Following low-stringency washes and a 2-day exposure, strongly hybridizing bands could be identified. One degenerate oligonucleotide, SH87, was used as a hybridization probe to screen a sheep phage genomic library. Two independent phage contained the H-GADDDDD-OH coding sequence as part of a larger predicted protein. AP may originate as part of an intracellular precursor protein, with multistep processing leading to the release of the heptapeptide into mucosal secretions. There it may interact with other innate pulmonary defenses to prevent microbial infection.     

Procollagen III in bronchoalveolar lavage fluid of patients with allergic alveolitis and sarcoidosis

In 12 patients with sarcoidosis and 11 patients with allergic alveolitis concentration of procollagen III peptide in bronchoalveolar lavage fluid (in bronchial and alveolar fraction) was estimated using RIA method. In studied populations procollagen III levels were higher in comparison to control. In patients with allergic alveolitis and with DLCO < 60% pred. procollagen III peptide concentrations in BAL fluid were significantly higher than in patients with sarcoidosis. In patients with allergic alveolitis a positive correlation between BAL lymphocytes number and procollagen III peptide concentration was observed.     

Eosinophil chemotactic activity in bronchoalveolar lavage fluid obtained from Toxocara canis-infected rats

Angioplasty of the internal mammary artery (IMA) bypass graft has been shown to be a safe and effective revascularization procedure. However, angiographic and long term clinical outcomes in the high-risk group of patients presenting with rest angina has not been well documented. We report the results of IMA angioplasty in 20 patients with rest angina out of 614 (3.2%) who received a left IMA graft at our institution between April 1987 and September 1994. All patients were admitted with rest angina, 12 patients demonstrated persistent ischemia despite medical therapy, two patients were in heart failure, and one patient was in cardiogenic shock. Balloon angioplasty was successful in 15 of 20 patients (75%). Failed angioplasty was associated with either severe IMA tortuousity (three patients) or inability to cross the anastomotic stenosis with the guide wire (two patients). Each of these five patients required angioplasty of either the native left anterior descending artery or other saphenous vein grafts for clinical stabilization. No patient suffered a major complication (myocardial infarction, emergent coronary bypass surgery, death). Clinical follow-up was obtained in all 20 patients (6 months, 7 years, mean 27 months). Twelve patients (60%) were asymptomatic or had stable angina at follow-up, and 8 returned with anginal symptoms. Four patients required repeat angioplasty for disease in other vessels, two were treated medically for angina, one underwent repeat CABG, and cardiac transplantation was performed in one patient for refractory heart failure. Angiographic follow up was obtained in 10/15 (66%) successful angioplasty patients, and only one patient demonstrated restenosis at the treated site (10%). During follow up one patient developed an IMA stenosis at a previous dissection site in the body of the graft that was treated with angioplasty. These results suggest that IMA angioplasty in patients with rest angina is associated with excellent long term patency and clinical efficacy, as well as low procedural risk.     

Distribution of asbestos bodies in the human lung as determined by bronchoalveolar lavage

Asbestos-related lung diseases tend to have distinct local distributions, for example, asbestosis first appears and tends to be more severe in the peripheral parts of the lower lung zones. The risk for asbestosis is related to the total asbestos burden of the lung. This suggests that the lower lobes in asbestos-exposed individuals may contain more asbestos than the other lobes. To test whether such topographic differences exist, we compared the number of retrieved asbestos bodies (AB) per ml BAL fluid in three groups of occupationally asbestos-exposed subjects who underwent BAL at different sampling sites. In Group 1 (n = 24) we performed BAL at three sites, namely in a segment of the right upper, right middle, and right lower lobe, to evaluate differences in asbestos body burden from lung apex to basis. There was a distinct increase in BAL asbestos body concentrations from the upper (21.2 +/- 9.1 AB/ml BAL fluid) to the middle (30.4 +/- 12.8 AB/ml BAL fluid) and to the lower lobe (56.0 +/- 20.2 AB/ml BAL fluid), all differences being significant (p < 0.01). In Group 2 (n = 40), we found good interlobar correlations for asbestos body counts between the right middle lobe (21.0 +/- 5.8 AB/ml BAL fluid) and the lingula (22.4 +/- 5.9 AB/ml BAL fluid) (r = 0.941, p < 0.001) and, in Group 3 (n = 15), between the ventral basal segment of the right (41.2 +/- 13.6 AB/ml BAL fluid) and left lung (39.0 +/- 13.6 AB/ml BAL fluid) (r = 0.966, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of annexin I in bronchoalveolar lavage fluid from patients with cystic fibrosis

BACKGROUND/AIMS: The number of perisinusoidal myofibroblasts has been shown to be increased in hepatocellular carcinoma, as compared to cirrhosis. This increase might suggest a cooperative relationship between tumour cells and myofibroblasts. To assess this relationship, we undertook: (a) an immunohistochemical study to confirm the existence of an increased number of perisinusoidal myofibroblasts in human hepatocellular carcinoma, as compared to cirrhosis with or without liver cell dysplasia, (b) an in vitro study testing the role of normal or tumoral human hepatocytes in myofibroblast proliferation. METHODS: Forty explanted cirrhotic livers, including 14 with hepatocellular carcinoma and 24 with liver cell dysplasia, were studied. Myofibroblasts were detected by immunohistochemistry using an antibody directed against alpha-smooth muscle actin. Hepatic myofibroblasts in culture were obtained by outgrowth from human liver explants. RESULTS: There was a progressive increase in the number of perisinusoidal myofibroblasts, from cirrhotic nodules without dysplasia to liver cell dysplasia and hepatocellular carcinoma. Conditioned medium from isolated normal human hepatocytes had only minor mitogenic effects on myofibroblasts, as assessed by measuring DNA synthesis and cell growth. In contrast, conditioned medium from a human hepatoma cell line (HepG2 cells) markedly stimulated the proliferation of human myofibroblasts. This mitogenic activity was stored in HepG2 cells and secreted in the extracellular medium rather than being simply released following cell lysis. CONCLUSIONS: These results suggest that the increased number of myofibroblasts in hepatocellular carcinoma might be due to a paracrine mechanism involving soluble mitogenic factor(s) secreted by tumour cells.     

Elevated levels of cytokeratin 19 in the bronchoalveolar lavage fluid of patients with chronic airway inflammatory diseases--a specific marker for bronchial epithelial injury

Cytokeratin 19 (CK19) is a specific cytoskeletal structure of simple epithelia, including bronchial epithelial cells (BEC). Since CK19 is released from injured bronchial epithelium, we investigated the levels of CK19 fragments in the bronchoalveolar lavage fluid (BALF) of eight patients with chronic airway inflammatory diseases (CAID) using an enzyme-linked immunosorbent assay (ELISA). Included in our test group were four cases of chronic bronchitis, three cases of bronchiectasis, and one case of diffuse panbronchiolitis. There were also 15 control subjects (five asymptomatic smokers and 10 nonsmokers). BALF from the nonsmokers as well as from the asymptomatic smokers contained few CK19 fragments (0.2 +/- 0.2 and 1.9 +/- 0.8 pg/ml, respectively). In contrast, significantly high levels of CK19 were present in the BALF of patients with CAID (21.7 +/- 5.7 pg/ml; p < 0.01 versus nonsmoking controls). In addition, CK19 fragment concentrations in BALF correlated significantly with the number of neutrophils (r = 0.722, p < 0.01) but not with the numbers of macrophages or lymphocytes in BALF. BALF from patients with CAID contained high levels of neutrophil elastase (NE) activity, suggesting that NE might be an important stimulus for the release of CK19 from BEC. To prove this, we incubated BET-1A cells, a human immortalized bronchial epithelial cell line, both in the absence and the presence of inflammatory mediators (including NE, tumor necrosis factor-alpha [TNF-alpha], and hydrogen peroxide). We then measured the concentration of CK19 fragments in the culture supernatants with ELISA. BET-1A cells released CK19 fragments into their culture supernatants after treatment with NE but not after treatment with TNF or hydrogen peroxide. Further, we demonstrated that CK19 cleaved by NE could not be detected by ELISA. Our results suggest that CK19 measurement in BALF is useful for assessing the presence of bronchial epithelial injuries.