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.     

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.     

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.     

Neutrophil chemotactic activity in bronchoalveolar lavage fluid of patients with AIDS-associated Pneumocystis carinii pneumonia

Of the various classes of human genetic disorders, aneuploidy is the most prevalent. Besides its association with maternal age and its predominant origin during maternal meiosis I, little is known about the etiology of aneuploidy. Although various classes of chemicals have been shown to induce aneuploidy in experimental systems, there is no definitive evidence for the role of chemically induced aneuploidy and adverse human health effects, particularly germ cell effects. Thus, it is important to understand the potential of chemicals for inducing aneuploidy in germ cells. There are conflicting data in the literature about the ability of thiabendazole (TBZ) to induce aneuploidy; therefore, we investigated the potential of TBZ for inducing aneuploidy in oocytes. Superovulated ICR female mice were administered 0, 50, 100, or 150 mg/kg TBZ by intraperitoneal injection. The frequencies and percentages of hyperploid oocytes were 0/472 (0), 2/410 (0.5), 6/ 478 (1.3), and 3/427 (0.7) for control, 50, 100, and 150 mg/kg TBZ, respectively. The difference between controls and the 100 mg/kg dose was statistically significant. Also, the proportions of ovulatory mice and the number of oocytes collected per ovulatory female were reduced in the TBZ groups relative to controls. Based on these results, we conclude that TBZ induces a small, but significant increase in the frequency of aneuploid oocytes at toxic doses that also impair ovulation.     

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.     

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.     

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.     

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.     

High levels of elastolytic activity in bronchoalveolar lavage fluid from a patient with idiopathic interstitial pneumonia]

A 63-year-old female with acure exacerbution of idiopathic interstitial pneumonia (IIP) showed high levels of elastolytic activity in bronchoalveolar lavage fluid (BALF). She was admitted to our hospital with progressive dyspnea. Arterial blood gas analysis and pulmonary function tests were abnormal. Examination of the bronchoalveolar lavage fluid revealed an increased numbers of neutrophils. Despite the administration of corticosteroids, the patient died of respiratory failure. A high level of elastolytic activity was present in the BALF. Western immunoblot analysis, using an anti-alpha 1-protease inhibitor (PI) antibody, revealed a truncated alpha 1-PI in the BALF. These findings suggest that an imbalance between protease and PI in the lower respiratory tract contribute to lung tissue damage in patients with IIP.     

Utility of routine testing of bronchoalveolar lavage fluid for cryptococcal antigen

All cryptococcal antigen (CrAg) testing performed at our institution between 1989 and 1994 was reviewed for utility of routinely testing of bronchoalveolar lavage fluid (BAL) for this antigen. Forty-two of 1,506 BAL specimens were positive. Seventeen of these were felt to represent false positives (sensitivity, 71%; positive predictive value, 0.59). The data on CrAg in cerebrospinal fluid and serum and the fungal culture and histological results of BAL specimens did not support continued, routine testing of BALs for CrAg to diagnose cryptococcosis.     

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.     

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.     

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.     

Studies of lymphocyte subpopulations of bronchoalveolar lavage fluid in HTLV-I seropositive patients with uveitis

To determine whether HTLV-I infection is associated with uveitis, we investigated the bronchoalveolar lavage fluid (BALF) cells in 23 patients with uveitis. Of these, sarcoidosis was diagnosed in 13 patients, but could not be confirmed in the remaining 10 patients (non-sarcoidosis). Three of 13 patients (23.1%) with sarcoidosis and 7 of 10 non-sarcoidosis patients (70.0%) were HTLV-I seropositive. In BALF, no significant differences were observed between the HTLV-I seropositive and seronegative patients. However, in non-sarcoidosis patients, total cell counts, CD3+ and CD+ HLA-DR+ cells in BALF were significantly higher in seropositive patients than in seronegative patients. CD3+ CD25+ cells in BALF were markedly increased in 3 of 7 non-sarcoidosis seronegative patients. These findings indicated that BALF lymphocytes in HTLV-I seropositive non-sarcoidosis patients were more activated than those in seronegative non-sarcoidosis patients, supporting the hypothesis that HTLV-I infection may be associated with uveitis.     

Measurement of T cell subsets in bronchoalveolar lavage fluid for diagnosis of pulmonary sarcoidosis

Pulmonary sarcoidosis is characterized by the accumulation in the lower respiratory tract of large numbers of activated CD4 T cells and elevated ratio of CD4/CD8 in bronchoalveolar lavage fluid (BALF). To study the value of CD4/CD8 ratio in BALF in the diagnosis of pulmonary sarcoidosis, we measured T cell subsets in BALF of patients with pulmonary sarcoidosis. The CD4/CD8 ratio in the patients (7.5 +/- 4.3) was significantly higher than that of the controls (2.1 +/- 0.7). The sensitivity and specificity of CD4/CD8 ratio in BALF for the diagnosis of sarcoidosis were 86% and 100%, respectively. We found that the CD4/CD8 ratio in BALF plays an important role in the diagnosis of pulmonary sarcoidosis. The analysis of CD4 in BALF may be useful in assessing the activity of sarcoidosis. The measurement of the CD4/CD8 ratio may be used to determine prognosis of pulmonary sarcoidosis.     

Pulmonary alveolar proteinosis: diagnosis using routinely processed smears of bronchoalveolar lavage fluid

AIMS: For the diagnosis of pulmonary alveolar proteinosis from bronchoalveolar lavage specimens it is normally necessary to make an ultrastructural examination. However, this is thought to be impractical for bronchoalveolar lavage specimens that have been routinely fixed in ethanol. In the present study, bronchoalveolar lavage cytology smears on slide glasses were examined directly ultrastructurally to make a diagnosis of pulmonary alveolar proteinosis. METHODS: Bronchoalveolar lavage smears from three pulmonary alveolar proteinosis patients were stained with Papanicolaou and periodic acid-Schiff (PAS) for identification of amorphous globular structures. Subsequently, they were refixed with glutaraldehyde and osmium tetroxide, and embedded in epoxy resin. Ultrathin sections were cut and examined ultrastructurally. RESULTS: Papanicolaou stained specimens from pulmonary alveolar proteinosis patients contained scattered amorphous or granular globules, 20-50 microns in diameter, which were PAS positive. Ultrastructural examination of the globules revealed multilamellated structures, characteristic of pulmonary alveolar proteinosis, in all cases. CONCLUSIONS: In general, it is thought that the morphological diagnosis of pulmonary alveolar proteinosis from bronchoalveolar lavage specimens requires both cytological and ultrastructural examination. However, the amorphous globules evident on cytology smears proved to contain multilamellated structures so that they can themselves be used as diagnostic evidence.