Increasing evidence suggests a role for the activation of the coagulation cascade in the pathogenesis of PF. In patients with IPF and PF secondary to systemic sclerosis, TF expression is upregulated on type II pneumocytes . In the latter group of patients, thrombin is increased in bronchoalveolarlavage fluid (BALF) . Further evidence for a role of the activation of blood coagulation stems from the observation that anticoagulants attenuate collagen deposition in a rat model of bleomycin-induced lung fibrosis . From a clinical point of view, IPF patients are at increased risk for pulmonary thromboembolism . Finally, a small, open label clinical trial has shown an increased 3-year survival in patients treated with anticoagulants (either warfarin or low molecular weight heparin) plus prednisone compared to the prednisone alone group , although a more recent, randomized trial with warfarin and no prednisone failed to confirm the result .
clinical operations . The study was approved by the ethics committee of Nanjing Chest Hospital. All the clinical works fulfilled the guideline of the bronchoscopy treatment towards the pulmonary tuberculosis [18,19]. As a kind of invasive operation, bronchoscopy requires strictly trained operator and the formal consent from every patient before being undergone. In clinic, every enrolled patient must sign a notification which declaims the aims, methods, merits and risks of this operation in detail. On the base of patients’ full understanding and when the signatures were obtained, the bronchoscopy was allowed to be undergone and the bronchoal- veolar lavage fluid was obtained. If the bronchoalveolarlavage fluid was suit for our research work, the corresponding patient would be further informed that his/her sample would be fur- ther used in research work after the relative clinical works were done. In this step, after obtain- ing the verbal consent from the patient, our research would be undertaken. Our lab work was started after the samples being discarded. This study didn't involve patient private information and any adjustment or difference in clinical treatment. Therefore, all the samples were not in- tended to be collected specially for the research but a step after the routine clinical exam. We provided the scanning file of the pre-operation notification (original manuscript in Chinese used in clinical work) and the translated file made by authorized translator (manuscript in En- glish) in supporting information (S1 and S2 Files).
Increased numbers and altered activities of pulmonary inflam- matory cells as well as enhanced elastolysis are a common feature of COPD. Factors like neutrophil elastase (NE) or matrix metalloproteases (MMP) in bronchoalveolarlavage (BAL) or sputum, are considered as markers for degradation and repair processes . Among the best-studied systemic markers in COPD are the acute phase protein CRP (C-reactive protein) and fibrinogen, which was recently shown to be the most repeatable marker in a large panel study of serum markers analyzed in the ECLIPSE study . Serum CRP is associated with mortality, morbidity, number of exacerbations, and inversely related to lung function indices [13,14]. Systemic inflammation is also reflected by increased serum concentrations of IL-6, TNFa and MCP-1 in COPD patients [1,15]. Serum IL-6 and CRP moderately correlate, are fairly stable over 1 year  and are increased in COPD patients with metabolic syndrome . However, the extent to which serum markers mirror ongoing inflammatory processes within the lung is largely unknown. Recently published data from the ECLIPSE study showed only a weak association between sputum neutrophils and 4 serum markers . While there is a lot of data available on potential biomarkers for COPD, only a few studies have addressed the issue of repeatability of multiple systemic and pulmonary markers [12,16,18,19].
Paracoccidioidomycosis (PCM) is an important systemic fungal disease, particularly among individuals living and working in rural areas of endemicity in Latin America, who, without antifungal therapy, may develop fatal acute or chronic infection. For such patients, the detection of antibody responses by immunodiffusion is of limited value due to false-negative results. In contrast, the detection of Paracoccidioides brasiliensis gp43 circulating antigen may represent a more practical approach to the rapid diagnosis of the disease. Accordingly, an inhibition enzyme-linked immunosorbent assay (inh-ELISA) was developed for the detection of a 43-kDa P. brasiliensis -specific epitope incorporating a species-specific murine monoclonal antibody. With sera from patients with acute and chronic forms of the disease (n ⴝ 81), the overall sensitivity of the test was found to be 95.1%, while specificity was found to be 97.5% compared to that with normal human sera from blood donors (n ⴝ 93) and sera from patients with other chronic fungal infections (histoplasmosis [n ⴝ 33] and crypto- coccosis [n ⴝ 20]). The inh-ELISA detected circulating antigen in 100% of patients with the acute form of PCM and in 95.31 and 100% of patients with the chronic multifocal and unifocal forms of PCM according to the patient’s clinical presentation. Cerebrospinal fluid from 14 patients with neuroparacoccidioidomycosis and 13 samples of bronchoalveolarlavage fluid from patients with pulmonary unifocal PCM were also tested for gp43 detection, with the test showing 100% sensitivity and specificity. This novel, highly specific inh-ELISA repre- sents a significant addition to the existing tests for the diagnosis of PCM.
blinded fashion by an infectious diseases specialist or a chest specialist, and by a radiologist. Briefly, five criteria for clinical diagnosis of TB were used: 1) patients with clinical symptoms and signs suggestive of TB diseases (such as sustained cough, weight loss, fever), chest X-rays that were typical of TB (typically showing a tuberculosis-like lesion), or a clinical response associated with administration of anti- tuberculous drugs, in the absence of other antimicrobial agents (clinical response was defined as improvement of chest X-ray/clinical symptoms); 2) clinical samples from Fig. 1 - Flowchart for the categorization of patients into different diagnostic groups. TB, tuberculosis; BAL, bronchoalveolarlavage; DTB, direct detection assay.
The purpose of the present study was to validate the quantitative culture and cellularity of bronchoalveolarlavage (BAL) for the diag- nosis of ventilator-associated pneumonia (VAP). A prospective vali- dation test trial was carried out between 1992 and 1997 in a general adult intensive care unit of a teaching hospital. Thirty-seven patients on mechanical ventilation with suspected VAP who died at most three days after a BAL diagnostic procedure were submitted to a postmor- tem lung biopsy. BAL effluent was submitted to Gram staining, quantitative culture and cellularity count. Postmortem lung tissue quantitative culture and histopathological findings were considered to be the gold standard exams for VAP diagnosis. According to these criteria, 20 patients (54%) were diagnosed as having VAP and 17 (46%) as not having the condition. Quantitative culture of BAL effluent showed 90% sensitivity (18/20), 94.1% specificity (16/17), 94.7% positive predictive value and 88.8% negative predictive value. Fever and leukocytosis were useless for VAP diagnosis. Gram stain- ing of BAL effluent was negative in 94.1% of the patients without VAP (16/17). Regarding the total cellularity of BAL, a cut-off point of 400,000 cells/ml showed a specificity of 94.1% (16/17), and a cut-off point of 50% of BAL neutrophils showed a sensitivity of 90% (19/20). In conclusion, BAL quantitative culture, Gram staining and cellularity might be useful in the diagnostic investigation of VAP.
This paper describes the in vivo Bronchoalveolarlavage (BAL) technique by endoscopy in tapirs (Tapirus terrestris) with clinical signs of tuberculosis. The technique was performed in two tapirs, male and female, from Curitiba Zoo, Paraná, Brazil. A flexible endoscope and a polyethylene catheter were used after the chemical restraint of the animals. For BAL technique, 60mL of saline 0.9% were infused with a polyethylene catheter, introduced by the endoscope’s working channel, and 15mL of BAL were recovered, analyzed and submitted to cytocentrifugation. Slides were stained by Papanicolaou, periodic acid-Schiff (PAS) and Ziehl-Neelsen methods contained high quantity of inflammatory cells on light microscopy (macrophages 27.5%, lymphocytes 0.5%, neutrophis 67% and eosinophis 5%). BAL samples were submitted to culture, bacilloscopy and PCR and were negative for both animals. Based on this study, it was concluded that the bronchoalveolarlavage technique in tapirs is feasible, simple, noninvasive, practical and fast, providing an important clinical information in vivo regarding the functional status of the lower respiratory tract.
The sample was considered satisfactory if it had less than ten squamous epithelial cells, more than 25 leukocytes per 100x microscopic field and the presence of alveolar macrophages . Samples that did not have these characteristics were considered unsatisfactory and the sputum induction was repeated within 48 hours of the first procedure. BronchoalveolarLavage Processing
Bronchoalveolarlavage (BAL) is a procedure that retrieves cells and other elements from the lungs for evaluation, which helps in the diagnosis of pulmonary diseases. The aim of this study was to perform this procedure for cellular analysis of BAL fluid alterations during experimental infection with Aelurostrongylus abstrusus in cats. Fourteen cats were individually inoculated with 800 third stage larvae of A. abstrusus and five non-infected cats lined as a control group. The BAL procedure was performed through the use of an endotracheal tube on the nineteen cats with a mean age of 18 months, on 0, 30, 60, 90, 120, 180 and 270 days after infection. Absolute cell counts in the infected cats revealed that alveolar macrophages and eosinophils were the predominant cells following infection. This study shows that the technique allows us to retrieve cells and first stage larvae what provides information about the inflammatory process caused by aelurostrongylosis. INDEX TERMS: Cytology, immunology, Aelurostrongylus abstrusus, aelurostrongylosis, cats.
The rats were euthanized by anesthetizing with ether inhalation and exsanguinated by cardiac puncture. Briefly, the thorax was opened by a median incision, the left bronchus was clamped with forceps, and a small incision was made in the the right bronchus and a plastic catheter attached to a 5-mL syringe was inserted. Bronchoalveolarlavage was performed a total of two times by repeated flushing with 5 ml ice-cold PBS, with gentle massage of the lungs. The bronchoalveolarlavage fluids were pooled and stored on ice until further use (10).
Paracoccidioidomycosis (PCM) is a systemic infection caused by the fungus Paracoccidioides brasiliensis and is believed to be the leading cause of fungal pulmonary infection. In this study, we used an inhibition enzyme-linked immunosorbent assay to diagnose pulmonary PCM based on the detection of 43-kDa and 70-kDa molecules in bronchoalveolarlavage fluids. The results were compared with results obtained by classical methods for antibody detection.
The present study used an alternative method for safe DNA extraction from bronchoalveolarlavage, and this protocol may reduce the time required to obtain definitive diagnostic results, respecting the presumptive diagnosis of melioidosis, which correctly identified B. pseudomallei even before the confirmation by culturing, which occurred after 5 days. Also, it is easily applicable for routine diagnostic purposes in many laboratories in developing countries, which have our reality.
Bronchoalveolarlavage fluid (BALF) viral titers were measured for each lamb by flushing the excised right caudal lung lobe with 5 ml of cold modified Iscove’s media (42.5% Iscove’s modified Dulbecco’s medium, 7.5% glycerol, 1% heat-inactivated FBS, 49% DMEM, and 5 m g/ml kanamycin sulfate) after which 1 ml of the resulting BAL fluid was placed on ice for immediate use in our standard infectious focus assay. For this, 1 ml BALF sample collected from each lamb was applied to HEp-2 cells grown to 70% confluence in 12-well culture plates (Fisher Scientific, Hanover, IL) in DMEM media (Mediatech, Inc., Manassas, VA) supplemented to 10% with heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA) and 50 m g/ml kanamycin sulfate (Invitrogen). Each sample was analyzed at full-strength and at four additional serial-dilutions of 1:10, 1:100, 1:1,000 and 1:10,000. 200 m l of each diluted BALF sample was assessed in duplicate. Overnight primary antibody (MAb to RSV Fusion Protein, Clone: RSV 3216 (B016), Meridian Bioscience, Cincin- nati, OH) incubation was followed by washing and secondary antibody (Goat anti-Mouse Fab’ conjugated to AlexaFluor 488, Invitrogen) incubation for 30 minutes. Plates were rinsed and inspected for the presence of fluorescing foci of infection using the FITC/GFP filter on an inverted fluorescence microscope (Olym- pus CKX41, Center Valley, PA). Five or more fluorescing cells were counted as single focal events. Aliquots of BALF in PBS from accessory lobes were assessed for total nucleated cell count and cytology. Counts were performed using the ADVIA 120 TM automated hematology analyzer (samples were diluted 1:2 in isotonic saline prior to cell counting). Cytospin preparations of BALF were created using a Shandon Cytospin 3. Modified Wright’s was used to stain the slides and differential cell counts (based on a 300 cell differential) were performed by a board certified veterinary clinical pathologist blinded to the identity of all samples.
Regarding the analysis of bronchoalveolarlavage, the first sample was collected 35.00 ± 13.84 minutes after in- tubation (mean ± SD), and the mean volume infused was 67.69 ± 17.39 mL with 29.77% recovery of total volume. The second sample was collected 43.23 ± 22.58 minutes after the end of CB, and 69.23 ± 19.35 mL were infused and 25.1% were recovered. Plasma and BAL levels of cytokines are pre- sented in Table II. Data regarding blood oxygenation and lung compliance are presented in Table III; non-measured levels of compliance correspond to the moments patients were ex- tubated.
Pulmonary coagulopathy is intrinsic to pulmonary injury including pneumonia. Anticoagulant strategies could benefit patients with pneumonia, but systemic administration of anticoagu- lant agents may lead to suboptimal local levels and may cause systemic hemorrhage. We hypothesized nebulization to provide a safer and more effective route for local administration of anticoagulants. Therefore, we aimed to examine feasibility and safety of nebulization of recombinant human tissue factor pathway inhibitor (rh-TFPI) in a well-established rat model of Streptococcus (S.) pneumoniae pneumonia. Thirty minutes before and every 6 hours after intratracheal instillation of S. pneumonia causing pneumonia, rats were subjected to local treatment with rh-TFPI or placebo, and sacrificed after 42 hours. Pneumonia was asso- ciated with local as well as systemic activation of coagulation. Nebulization of rh-TFPI re- sulted in high levels of rh-TFPI in bronchoalveolarlavage fluid, which was accompanied by an attenuation of pulmonary coagulation. Systemic rh-TFPI levels remained undetectable, and systemic TFPI activity and systemic coagulation were not affected. Histopathology re- vealed no bleeding in the lungs. We conclude that nebulization of rh-TFPI seems feasible and safe; local anticoagulant treatment with rh-TFPI attenuates pulmonary coagulation, while not affecting systemic coagulation in a rat model of S. pneumoniae pneumonia.
‘‘100’’ form, compared to those with more SP-A present as less complex forms (Fig. 2 c, d). This association was seen both in BAL Figure 1. Illustration of the column characteristics for the separation of SP-A present in bronchoalveolarlavage or serum samples. Samples from different patients were run on the column and SP-A content of each fraction was determined and plotted, normalized to its total amount eluting from the column. Here, selected samples from three different subjects, each with a distinct extreme pattern of elution, are shown (patient A (squares), having mainly one initial peak around fraction 10 contains 18-mers and larger; patient B (triangles), having mainly a peak around fraction 15 with SP-A 6–12-mers; and patient C (circles), having mainly the lower molecular weight forms of SP-A).
The World Health Organization reports that 235 million people are currently affected by asthma. This disease is associated with an imbalance of Th1 and Th2 cells, which results in the upregulation of cytokines that promote chronic inflammation of the respiratory system. The inflammatory response causes airway obstruction and can ultimately result in death. In this study we evaluated the effect of 19-acetoxychavicol acetate (ACA) isolated from Alpinia galanga rhizomes in a mouse model of ovalbumin (OVA)-induced asthma. To generate the mouse model, BALB/c mice were sensitized by intraperitoneal injection of OVA and then challenged with OVA inhalation for 5 days. Mice in the vehicle control group were sensitized with OVA but not challenged with OVA. Treatment groups received dexamethasone, 25 mg/kg/day ACA, or 50 mg/kg/day ACA for 5 days. Asthma-related inflammation was assessed by bronchoalveolarlavage fluid cell counts and histopathological and immunohistochemical analysis of lung tissues. Our results showed that ACA reduced the infiltration of white blood cells (especially eosinophils) and the level of IgE in the lungs of mice challenged with OVA and suppressed histopathological changes such as airway remodeling, goblet-cell hyperplasia, eosinophil infiltration, and glycoprotein secretion. In addition, ACA inhibited expression of the Th2 cytokines interleukin (IL)-4 and IL-13, and Th1 cytokines IL-12a and interferon-c. Because asthmatic reactions are mediated by diverse immune and inflammatory pathways, ACA shows promise as an antiasthmatic drug candidate.
Cystic fibrosis (CF) is a genetic disease with many airway pathological features, including aberrant epithelial sodium channel (ENaC) function, persistent Pseudomonas aeruginosa (PA) infection and neutrophil-dominant inflammation. PA infection in CF airways is difficult to treat due to antibiotic resistance and other factors. Recently, α1-antitrypsin (A1AT) have been shown to be effective to reduce CF airway PA infection. However, there is a dearth of studies about the mechanisms underlying A1AT’s therapeutic effects. The goal of our study is to provide an animal model of A1AT therapy in CF lungs. ENaC transgenic mice with PA infection were used as a CF-like model. Mice were intratracheally treated with PA or saline (control) in a fibrin plug. Two hours after PA infection, aerosolized A1AT were delivered to mouse lungs once daily. At day 1 and day 3 post PA infection, lung inflammation, PA load as well as host defence protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) were measured. At day 1 post PA infection when A1AT was delivered once to ENaC trans- genic mouse lungs, A1AT did not reduce lung inflammation (e.g., neutrophils) and PA load. However, at day 3 post PA infection when ENaC transgenic mice received three repeated A1AT treatments, a significant decrease in airspace inflammation and PA load was observed. Although A1AT prevented the loss of SPLUNC1 in bronchoalveolarlavage fluid of PA-infected wild-type mice, it did not restore SPLUNC1 levels in ENaC transgenic mice. Our current study has provided a valid and quick A1AT therapeutic model in CF-like lungs that may serve as a platform for future mechanistic studies about how A1AT exerts benefi- cial effects in human CF patients.
hypoxemia, and hypercapnia. An increased (P < 0.05) cell influx was observed in bronchoalveolarlavage, with a predominance of neutro- phils. Total protein and MDA concentrations were also higher (P < 0.05) in the septic groups compared to control. A higher tumor necrosis factor-alpha (P < 0.05) concentration was also found in these animals. Changes in all parameters were more pronounced with the higher bacterial inoculum. PTX administered prior to sepsis reduced (P < 0.05) most functional alterations. These data show that an E. coli ip inoculum is a good model for the induction of lung dysfunction in sepsis, and suitable for studies of therapeutic interventions.