Depressed urokinase activity in bronchoalveolar lavage fluid from patients with sarcoidosis,silicosis or idiopathic pulmonary Rbrosis: relationship to disease severity

Intraalveolar fibrinolysis, is regulated by the concerted actions of plasmin, plasminogen activators (PAs), and their specific inhibitors (PAIs). This event is considered as a critical step in the pathogenesis of pulmonary fibrosis. The aim of this study was to evaluate whether local PA activity can be held as a marker of fibrosis in chronic interstitial lung disorders (ILD). Changes in both PA activity and PA-related proteins (urokinase-type PA (uPA), tissue-type PA (tPA), PAI-1 and PAI-2) were assessed in bronchoalveolar fluid (BALF) of 60 subjects: 18 healthy controls, 18 non-fibrotic sarcoidosis patients, 16 patients with idiopathic pulmonary fibrosis (IPF) and eight silicotic patients with established fibrosis. We observed a significant decrease of BALF PA activity in the three groups of patients as compared with controls. Reduction in BALF PA activity was compatible with lower uPA protein levels associated, especially in IPF patients, with an increased occurrence of PAI-1 and PAI-2 antigens. Soluble tPA antigen was never detected either in control subjects or in patients. Most importantly, the reduction in BALF PA activity and uPA protein levels was found to be most severe in patients with advanced fibrotic disease, namely IPF, while moderate and only weak alterations were found in silicosis and non-fibrotic sarcoidosis, respectively. In addition, significant positive correlations were found between BALF PA activity and functional impairment as assessed by TLC % and DL_CO%. Finally, the reduction in uPA and PA activity levels observed in BALF from sarcoidosis patients was found to be proportional to the degree of BAL lymphocytosis. These findings indicate that an intense reduction in BALF PA activity is associated with severe stages of the parenchymal disease, possibly reflecting the degree of the fibrotic process.     

Pulmonary histopathology and alteration in cellular pattern of bronchoalveolar lavage fluid in experimental silicosis

Pathogenesis of silicosis is still being evaluated. Cellular and histopathological changes in lung following acute and chronic exposure of quartz in rats have been investigated. Inbred wistar rats were given single intratracheal injection of quartz (10 mg in 0.05 ml saline) in groups of acute model, and inhalation of quartz (40 mg/m3 with air flow 5 l/hr in a simulation chamber, 6 hr/day) in groups of chronic model. The control groups were exposed to vehicles only. Rats were sacrificed on day 3, 5 and 7 of intratracheal injection and after 2, 4 and 8 weeks of inhalation. Total and differential cell counts (TC and DC) were performed in bronchoalveolar lavage fluid (BALF). Histopathology was done in the lungs. There was significant (P < 0.001) increase in TC and significant (P < 0.001) changes in percentage of inflammatory cell counts on DC in the BALF of silicotic rats. Histopathology showed progressive inflammatory and fibrotic response in quartz exposed lungs in both acute and chronic models. The results indicate duration dependent inflammatory changes in lungs of both the models. Changes in cell counts precede the histopathological changes and may serve as early biological marker for detection of silicosis.     

Hyaluronate in bronchoalveolar lavage fluid: a new marker in sarcoidosis reflecting pulmonary disease.

Hyaluronate (hyaluronic acid) was not detectable in bronchoalveolar lavage fluid from smoking or nonsmoking healthy volunteers but was present in fluid from 23 patients with sarcoidosis; the mean concentration was 16 micrograms/1 returned fluid (range less than or equal to 5-430) or, expressed in relation to the amount of albumin recovered, 0.22 micrograms/mg albumin (range less than or equal to 0.05-3.6). The serum hyaluronate concentrations in the patients with sarcoidosis were normal. There was a significant inverse correlation between vital lung capacity and hyaluronate concentrations in bronchoalveolar lavage fluid (p less than 0.001), and patients with abnormal lung volumes had hyaluronate concentrations that were on average six times higher than those in patients with normal vital capacity. Duration of disease, pulmonary radiological findings, and markers for macrophage activation (angiotensin converting enzyme) and lymphocyte activation (beta 2 microglobulin) were not correlated with bronchoalveolar lavage fluid hyaluronate. It was concluded that in sarcoidosis release of hyaluronate into the airways is related to lung volume and therefore to the course of the disease. Increased synthesis of hyaluronate in lung parenchyma may reflect activation of fibroblasts, and measurements of hyaluronate may have clinical value for prognosis and treatment.     

Hemosiderin-laden macrophages in bronchoalveolar lavage fluid.

Diffuse pulmonary hemorrhage syndromes occasionally present diagnostic problems if the clinical picture is not very typical. The presence of hemosiderin-laden alveolar macrophages in bronchoalveolar lavage (BAL) fluid is a useful diagnostic criterion for this entity. However, in many diffuse interstitial pulmonary diseases (DIPD) there is a lesion at the alveolocapillary barrier, and an exit of red blood cells could occur from the blood vessels, leading to the appearance of siderophages. The aim of this work was dual: to evaluate the presence and number of siderophages in different types of interstitial pulmonary disease and to compare the diagnostic yield of two ways of quantifying hemosiderin-laden macrophages. Three groups of patients--controls (n = 5), DIPD (n = 32) and diffuse pulmonary hemorrhage (n = 3)--were subjected to BAL, and a differential count was made on cytocentrifuged Diff-Quik- and Perl-stained preparations. On the latter, two different measurements were made: the number of macrophages laden with hemosiderin and a quantitative "score." The results of a conventional count (percent of Perl-positive macrophages) showed significant differences between the three groups considered overall. Applying a cutoff value of 20%, the sensitivity of this method was 100% and the specificity, 91.6%. The results of the hemosiderin score showed significant differences between the three groups. Applying a value of 50 as a cutoff, the sensitivity and specificity of the method were 100%. In control patients and carriers of DIPD, the percentage of alveolar macrophages was higher than in healthy subjects. Quantification of the hemosiderin content of alveolar macrophages improved the specificity of the diagnosis of diffuse pulmonary hemorrhage by BAL.     

Study of bronchoalveolar lavage fluid in paracoccidioidomycosis: cytopathology and alveolar macrophage function in response to gamma interferon; comparison with blood monocytes

Patients with paracoccidioidomycosis (PCM) present marked involvement of the lungs during the course of the mycosis. The purpose of this work was to obtain bronchoalveolar lavage (BAL) fluid from these patients to study the cytopathology, TNF levels and the oxidative and fungicidal response of alveolar macrophages (AMs) to in vitro incubation with recombinant IFN-gamma. To compare the lung and blood compartments, these determinations were also made in plasma and blood monocytes (BMs) obtained from the same patients. The cytopathology of BAL fluid revealed a predominance of macrophages, but with the presence of neutrophil exudation, and rare lymphocytes and epithelioid and giant cells. Comparison of the oxidative status and fungicidal activity of AMs and circulating BMs demonstrated that both cell types are highly activated for these two functions when compared to control cells. However, TNF levels were higher in BAL fluid than in plasma. The possible mechanisms involved in the hyperresponsiveness of cells from PCM patients are discussed.     

Proteomics of bronchoalveolar lavage fluid

Lung diseases are essentially multi-factorial diseases that require a global analysis, and thus, cannot be understood through the sole analysis of individual or small numbers of genes. Proteome analysis has rapidly developed in the post-genome era and is now widely accepted as the obligated complementary technology for genetic profiling. It has been shown to be a powerful tool for the study of human diseases and for identifying novel prognostic, diagnostic and therapeutic markers. During last years, proteomic approaches applied to lung diseases are centred on the analysis of proteins in samples, such as cell cultures, biopsies and physiological fluids like serum and, especially, bronchoalveolar lavage fluid (BALF). BALF is presently the most common way of sampling the components of the epithelial lining fluid (ELF) and the most faithful reflect of the protein composition of the pulmonary airways. This review focuses on the state of the investigations of BALF proteome and its powerful contribution both to a better knowledge of the lung structure at the molecular level and to the study of lung disorders at the clinical level.     

Galactomannan detection in bronchoalveolar lavage fluid

Prompt diagnosis of invasive pulmonary aspergillosis (IPA) remains a challenge. Galactomannan (GM) assay in serum has been incorporated into diagnostic criteria for IPA, but its performance varies depending on the population in which the test is used. GM assay on bronchoalveolar lavage (BAL) fluid aims to improve upon the test by applying it directly on samples from the target organ. The studies that have examined the utility of BAL-GM are a heterogeneous group, but the results are intriguing, especially in patients who are at risk for IPA from causes other than hematologic malignancy and neutropenia. BAL-GM had sensitivities ranging from 60% to 100% in this group, often far exceeding the performance of serum GM assay. The test shows promise as a useful adjunctive diagnostic modality in the diagnosis of IPA.     

Penicillium marneffei in bronchoalveolar lavage fluid

Penicillium marneffei is a rare human pathogen that may infect either healthy or immunocompromised hosts. In methenamine silver-stained preparations of bronchoalveolar lavage fluid from a patient with dermatomyositis on steroid treatment, round-to-oval intracellular and extracellular microorganisms were found. The finding of occasional septate and elongated forms established the microorganism as probably P marneffei, which was confirmed on culture. Distinguishing this rare fungus from Pneumocystis carinii is important because these two diseases require different forms of treatment.     

Database of bronchoalveolar lavage fluid proteins

Bronchoalveolar lavage during fiberoptic bronchoscopy is extensively used for investigating cellular and biochemical alterations of the epithelial lining fluid in various lung disorders. Two-dimensional electrophoresis (2-DE) offers the possibility to simultaneously display and analyze proteins contained in bronchoalveolar lavage fluid (BALF). We present the current status of 2-DE of BALF samples with an updated listing of the proteins already identified and of their level and/or posttranslational alterations in lung disorders. Alternatives to 2-DE of BALF samples and future prospects of proteomics to unravel lung functions and pathologies are discussed.     

Interleukin-2-administration intravenously and intrapleurally in a patient with primary pulmonary adenocarcinoma. Cellular responses in peripheral blood,intrapleural fluid and bronchoalveolar lavage

A case report of a patient who suffered from a rapidly progressive lung adenocarcinoma with malignant pleural effusions, is given. The patient failed to respond on two series of conventional cytotoxic drug therapy (Carboplatin, Etoposid). Interleukin-2 (IL-2) treatment was first started as intrapleural instillations (3.0 million IU per day in 6 days). A clear clinical response was achieved with ceasing of the pleural effusion, and the overall disease became stable. In the peripheral blood, there was an increase of CD₄ positive lymphocytes that remained elevated after finishing the installation period. Both in bronchoalveolar lavage (BAL), and in the pleural fluid, there was a marked decrease of cells recovered, possibly due to an enhanced tissue attachment of activated cells. A second analysis with subtyping of lymphocytes in BAL was impossible due to the low cell number. In the pleural fluid, the fractions of CD₃ positive cells increased from 20 to 71% while the ratio between CD₄ and CD₈ remained persistently elevated at 6.11. Because of the disappearance of the pleural effusion, the patient was thereafter treated with IL-2 given as a continuous infusion (18 million IU per square-metre during 24 hours for 5 days). Hereby a more pronounced cell response was achieved in the peripheral blood. In contrast to the intrapleural treatment route, not only CD₄ positive cells, but also the numbers of natural killer cells (NK) increased. However this treatment was also associated with a much higher degree of side effects. It can be concluded from both intrapleural and intravenous IL-2 therapy, that a clinical and immunological response was achieved. As this type of tumour is well known to respond poorly to conventional therapy, treatment with biological modifiers such as IL-2 may offer an interesting alternative in the future.Abbreviations BAL bronchoalveolar lavage - K-2 Interleukin-2     

Th1/Th2 cytokine pattern in bronchoalveolar lavage fluid and induced sputum in pulmonary sarcoidosis

Background

Recent studies suggest that angiotensin-converting enzyme (ACE) inhibitors may have beneficial effects for patients at risk for some types of infections. We examined the effect of prior outpatient use of ACE inhibitors on mortality for patients hospitalized with community-acquired pneumonia.

Methods

A retrospective cohort study conducted at two tertiary teaching hospitals. Eligible subjects were admitted with a diagnosis of, had a chest x-ray consistent with, and had a discharge ICD-9 diagnosis of pneumonia. Subjects were excluded if they were "comfort measures only" or transferred from another acute care hospital. Subjects were considered to be on a medication if they were taking it at the time of presentation.

Results

Data was abstracted on 787 subjects at the two hospitals. Mortality was 9.2% at 30-days and 13.6% at 90-days. At presentation 52% of subjects were low risk, 34% were moderate risk, and 14% were high risk. In the multivariable conditional logistic regression analysis, after adjusting for potential confounders, the use of ACE inhibitors at presentation (odds ratio 0.44, 95% confidence interval 0.22–0.89) was significantly associated with 30-day mortality.

Conclusion

Prior outpatient use of an ACE inhibitor was associated with decreased mortality in patients hospitalized with community-acquired pneumonia despite their use being associated with comorbid illnesses likely to contribute to increased mortality. Confirmatory studies are needed, as well as research to determine the mechanism(s) of this protective effect.     

Diffuse alveolar damage. Morphologic features in bronchoalveolar lavage fluid

OBJECTIVE: To evaluate the overall cytologic characteristics of diffuse alveolar damage (DAD) in bronchoalveolar lavage (BAL) specimens in search of features that could be useful in cytologic diagnosis. STUDY DESIGN: We evaluated BAL samples from patients with DAD obtained simultaneously with transbronchial biopsies (n = 8) or open lung biopsies (n = 2) or within 24 hours of autopsy (n = 2). The material was processed routinely for cytologic and histologic evaluation. RESULTS: The smears were moderately to highly cellular. All cases had large numbers of alveolar macrophages and/or desquamated alveolar cells. The epithelial component displayed various degrees of nuclear atypia. Some epithelial clusters were three-dimensional, with peripheral cells showing clear cytoplasm, protruding outwards and resembling hobnails. Other aggregates appeared two-dimensional, as sheets of cells with flattened and dense cytoplasm (squamotized). Both types of cell clusters were often associated with dense, basophilic or amphophilic, amorphous extracellular material. Counterparts of all the cytologic features were observed in the histologic material, including atypia of the alveolar lining with hobnailing, squamotization, amorphous extracellular material and hyaline membranes. CONCLUSION: The cytologic features of BAL represent a constellation of alveolar cell injury. Based on these features, DAD can be correctly diagnosed or suggested in BAL samples in the appropriate clinical setting.     

Expression of Fas antigen and Fas ligand in bronchoalveolar lavage from silicosis patients

OBJECTIVE: To understand the role of apoptosis through Fas/Fas ligand (FasL) interaction in the pathogenesis of silicosis, we examined the expression of Fas antigen, FasL and apoptosis in bronchoalveolar lavage fluid lymphocytes obtained from patients with silicosis. MATERIALS AND METHODS: Ten patients with silicosis, and 10 healthy controls were studied. Non-adherent cells were separated and analysed by cytometry for the expression of Fas antigen, FasL, and the co-expression of Fas/FasL. By double staining, we studied the FasL expression on CD4, CD8, CD56 and CD45RO-positive cells. DNA fragmentation was investigated by the terminal deoxy(d) UTP nick end labelling (TUNEL) method. RESULTS: We have found Fas and FasL expression in silicosis patients to be significantly higher than those in healthy controls. Interestingly, 6-18% of lymphocytes from silicosis patients co-expressed Fas and FasL. In silicosis patients, FasL was highly expressed on CD4+, CD56+ and CD45RO+ bronchoalveolar lavage cells. Fas antigen expressing cells showed DNA fragmentation characteristic for apoptosis. CONCLUSION: FasL was significantly expressed on cytotoxic effector and memory cells. The Fas/FasL system is implicated in the inflammatory process observed in silicosis patients.     

Detection of Candida antigen in bronchoalveolar lavage fluid

While bronchoalveolar lavage is frequently performed to evaluate immunocompromised hosts for infection, the significance of rare yeasts found on the cytologic examination of lavage fluid is unclear. This study used the latex agglutination method to test lavage fluids for Candida antigen to assess its usefulness in distinguishing Candida pneumonia from Candida colonization of the respiratory tract or oral contamination of the lavage specimen. Ninety-seven specimens from 87 patients were categorized on the basis of historical, microbiologic, cytologic and serologic data. Bronchoalveolar lavage fluids were positive for Candida antigen in 0 of 20 specimens from normal controls, 0 of 14 specimens from patient controls, 5 (36%) of 14 specimens from patients with Pneumocystis carinii pneumonia, 0 of 5 specimens from patients with gastrointestinal candidiasis, 0 of 9 specimens contaminated by oral-derived yeasts, 2 (10%) of 19 specimens from patients with probable Candida colonization and 15 (94%) of 16 specimens from patients with clinical and laboratory evidence of Candida pneumonia. We conclude that this test assists in the differentiation of Candida pneumonia from other situations in which yeasts are recovered by bronchoalveolar lavage.     

Reactive type II pneumocytes in bronchoalveolar lavage fluid

OBJECTIVE: To evaluate the prevalence of reactive type II pneumocytes (RPII) in bronchoalveolar lavage (BAL) fluid samples obtained from patients with various pulmonary disorders. STUDY DESIGN: Consecutive BAL fluid samples were screened for the presence of RPII on May-Grünwald-Giemsa-stained cytocentrifuge preparations. BAL fluid samples with and without RPII were compared with regard to prevalence, associated clinical diagnoses and cytologic findings. RESULTS: RPII were generally large cells with a high nuclear:cytoplasmic ratio and deeply blue-stained, vacuolated cytoplasm. Most RPII occurred in cohesive cell groups, and the vacuoles tended to be confluent. Cytologic findings associated with RPII were foamy alveolar macrophages, activated lymphocytes and plasma cells. RPII were present in 94 (21.7%) of 433 included BAL fluid samples. The highest prevalences were noted in patients with systemic inflammatory response syndrome and alveolar hemorrhage. In addition, RPII tended to occur more frequently in ventilator-associated pneumonia, Pneumocystis carinii pneumonia, extrinsic allergic alveolitis and drug-induced pulmonary disorders. In contrast, RPII were not observed in BAL fluid samples obtained from patients with sarcoidosis. CONCLUSION: RPII were prevalent in about 20% of BAL fluid specimens. They were associated mainly with conditions of acute lung injury and not observed in sarcoidosis.     

Detection of Schistosoma mansoni in bronchoalveolar lavage fluid. A case report.

BACKGROUND: Bronchoalveolar lavage (BAL) is a useful tool in the diagnosis of bacterial, viral, fungal and parasitic pulmonary infections. There have been rare reports of parasitic infestations in bronchoalveolar lavage fluid. This is the first case report on detecting a Schistosoma ova in BAL fluid. CASE: A 40-year-old, Egyptian male presented with a fever and productive cough. He had a right pleural effusion and segmental collapse of the right lower lobe. BAL fluid showed several ova of Schistosoma mansoni and established the diagnosis of schistosomiasis. Abdominal ultrasound revealed mild hepatic cirrhosis. CONCLUSION: Schistosomiasis should be considered in the differential diagnosis of pulmonary problems in patients with disseminated disease in endemic areas.     

Study of pulmonary experimental paracoccidioidomycosis by analysis of bronchoalveolar lavage cells: Resistant vs. susceptible mice

Adult Swiss (susceptible) and BALB/c (non-susceptible) mice were inoculated by the intravenous route with 1 × 10⁶ yeast cells of Paracoccidioides brasiliensis, strain 18. Immunologic parameters, histopathology and features of the bronchoalveolar lavage (BAL) were evaluated at week 2, 4, 8 and 16 post-infection. The pulmonary infection was progressive in Swiss mice and regressive in BALB/c mice. The numbers of total cells, lymphocytes and polymorphonuclear neutrophils increased in BAL, as well as the percentages of giant cells, and CD4 and CD8 positive cells. The ultrastructural study of BAL cells revealed a predominance of macrophages and a frequency of 13.2% of type II pneumocytes. As the infection progressed, the number of fungal cells and spreading macrophages, as well as the stimulated release of H₂O₂ by macrophages, increased. The animals exhibited an exacerbation of the humoral immune response and a depression of cellular immunity during the infection. There was a good correlation between the intensity and the pattern of the pulmonary histopathology and the cellular findings in the BAL. The present model reproduces some anatomoclinical patterns of the human disease and shows that BAL may be a useful tool in monitoring the pulmonary infection caused by P. brasiliensis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Double staining of intracellular cytokine proteins and T-lymphocyte subsets. Evaluation of the method in blood and bronchoalveolar lavage fluid

An immunocytochemical staining method has been developed for simultaneous staining of both cell surface markers (CD4 and CD8) and intracellular cytokine proteins IFN-, IL-4 and IL-5. Cell surface molecules were visualized with alkaline phosphatase, which was developed by Fast Blue BB. Intracellular cytokine proteins were detected by amino-ethyl carbazole. We applied this technique to T cells from T-cell lines and T-cell clones, peripheral blood mononuclear cells and broncho-alveolar lavage fluid cells. Cells were used either unstimulated or stimulated for 4h with 1ng/ml PMA and 1g/ml ionomycin, which proved to be an optimal stimulus taking cytokine staining, cell recovery and cell viability into account. We studied peripheral blood mononuclear cells from healthy subjects and found that without in vitro stimulation on average 0.4% of the cells were IFN- positive cells. In unstimulated broncho-alveolar lavage fluid cells of the 2 allergic asthmatic subjects studied so far we found higher numbers of cytokine-positive cells (up to 22% of the lymphocytes being IL-4⁺ cells). By in vitro stimulation, the numbers of cytokine-positive peripheral blood mononuclear cells from the healthy subjects were increased to maximally 5% IFN-⁺ cells. In stimulated lavage fluid cells from allergic asthmatic subjects maximally 34% of the lymphocytes became IFN-⁺. We conclude that this method allows detection of intracellular cytokine proteins in both CD4⁺ and CD8⁺ T cells without the need for stimulating the cells in vitro. In vitro stimulation may change the cytokine profile detected.