Non-bronchoscopic Bronchoalveolar Lavage as a Refinement for Safely Obtaining High-quality Samples from Macaques

Nonbronchoscopic bronchoalveolar lavage (NB-BAL) is a minimally invasive diagnostic and research tool used to sample the cells of lower airways and alveoli without using a bronchoscope. Our study compared NB-BAL and bronchoscopic bronchoalveolar lavage (B-BAL) in terms of costs, cell yields, and the number of post-procedural complications in macaques. We also analyzed procedure times, BAL fluid volume yields, and vital signs in a subset of animals that underwent NB-BAL. Compared with the B-BAL technique, NB-BAL was less expensive to perform, with fewer complications, fewer animals requiring temporary or permanent cessation of BALs, and higher cell yields per mL of recovered saline. The average procedure time for NB-BAL was 6.8 ± 1.6 min, and the average NB-BAL lavage volume yield was 76 ± 9%. We found no significant differences in respiration rate before, during, or after NB-BAL but did find significant differences in heart rate and oxygen saturation (SpO₂). This study demonstrates that NB-BAL is a simple, cost-effective, and safe alternative to B-BAL that results in higher cell yields per mL, improved animal welfare, and fewer missed time points, and thus constitutes a refinement over the B-BAL in macaques.

Bronchoalveolar lavage (BAL) is a minimally invasive diagnostic and research tool used to retrieve cells, microbes, and biomarkers from the lower airways and alveoli. The technique is used in both human and veterinary medicine to aid in diagnosing respiratory tract diseases such as lower airway infections, neoplasia, pulmonary hemorrhage, hypereosinophilic syndromes, hypersensitivity pneumonitis, and environmental lung diseases. BAL is also used in research to investigate the immunologic response of the lower airways to induced or spontaneous diseases. BAL fluid can be analyzed in terms of flow cytometry, gene expression, antibody titers, inflammatory mediators, and the confirmation or monitoring of experimental infections via culture or polymerase chain reaction.BAL may be performed with or without the use of a bronchoscope. Bronchoscopic BAL (B-BAL) has several advantages, including visualization of the upper and lower airways and the capacity to select particular lung lobes for fluid instillation and retrieval. B-BAL is also cited as having a better ability to “wedge” the scope into a bronchus, creating a tight seal that may improve volume yield.⁷ However, B-BAL has several disadvantages, including high equipment cost, the need for trained bronchoscopists, and the difficulty of effective and efficient instrument sterilization. Similarly, B-BAL may not be possible in small human or veterinary patients, and visualization of airways and selection of a particular lung lobe may not be necessary for research purposes.Nonbronchoscopic BAL (NB-BAL) is typically performed by passing a small suction catheter through an endotracheal (ET) tube into the lower airways. This technique has been used in infants and domestic cats requiring small diameter ET tubes because until recently the smallest available bronchoscope with a lavage channel would obstruct the ET tube lumen in these subjects.⁶ Although advancements in endoscope technology have made fine diameter broncho-fiberscopes with lavage channels available, NB-BAL has several advantages over B-BAL, including lower cost, minimal needs for technical skill, and no need for bronchoscope equipment. In addition, the use of sterile, single-use catheters reduces the potential for cross-contamination between patients and samples.⁹Our group, the Oregon National Primate Research Center (ONPRC) Infectious Disease Resource (IDR), provides technical support to facilitate nonhuman primate (NHP) studies in areas such as immunology, infectious disease pathogenesis, and safety and efficacy testing of therapeutics and vaccines. BAL fluid contains abundant effector memory CD8⁺ T cells derived from an easily accessible mucosal site⁴ and is commonly requested by our investigators, with requests of 10 to 50 samples per workday. We initially used the ONPRC Surgical Services Unit (SSU) to obtain samples using B-BAL; this approach was expensive, frequently required medical intervention (supplemental oxygen, terbutaline and/or furosemide), and caused many animals to miss multiple BAL time points due to periprocedural complications. In addition, due to high procedure volumes and a limited number of bronchoscopes, equipment was commonly cleaned but not sterilized between animals. Therefore, we sought an inexpensive, simple BAL technique that promoted equipment sterility between animals, provided comparable results for investigators, and resulted in better clinical outcomes for study animals. We hypothesized that NB-BAL would provide comparable research results (cell yields) to B-BAL at a lower cost to our researchers. We also hypothesized that using the NB-BAL method would minimize complications as compared with the B-BAL method. Finally, we analyzed procedure times, BAL fluid volume yields, and vital signs in a subset of animals that underwent NB-BAL.     

How to express tumor markers in bronchoalveolar lavage

INTRODUCTION: Bronchoalveolar lavage (BAL) is a fundamental technique in the diagnosis of different respiratory diseases including lung cancer. Tumor marker values can be determined in the BAL fluid, but controversy still exists about how to express the results. OBJECTIVE: The aim of this study was to determine the best method of expressing tumor markers in BAL, either referring to total proteins or volume of fluid recovered. PATIENTS AND METHODS: A prospective, randomized, non-blind study was carried out. Seventy-six patients (72 men and 4 women) diagnosed with lung cancer and 17 subjects without respiratory disease were included. BAL was performed in all patients and the fluid retrieved was divided into two fractions: a bronchiolar fraction (F0) and an alveolar fraction (F1). Five tumor markers: cytokeratin fragment 19 (CYFRA 21-1), squamous cell carcinoma antigen (SCC), tissue polypeptide antigen (TPA), tissue polypeptide-specific antigen (TPS) and neuron-specific enolase (NSE) as well as total protein were measured in both fractions. The concentrations were expressed in relation to the volume of BAL fluid recovered (ng or mU/mL) and in milligrams of total protein of lavage fluid (ng or mU/mg TP). The SPSS 11.01 software was used for statistical analysis. Mann-Whitney U test and ROC curves were developed when significant differences were found. RESULTS: We found significant differences in the CYFRA 21-1 values in the two BAL fractions and in both ways of expressing its concentration; in SCC in F1 expressed in ng/mg TP; in TPA in F0 expressed in mU/mg TP; in TPS in both fractions expressed in mU/mg TP, and in NSE in both fractions in ng/mg TP. The markers that best differentiated tumors from controls (ROC curves) were CYFRA 21-1 in F0 and NSE in both fractions in ng/mg TP. CONCLUSIONS: Our study demonstrates that the concentrations of tumor markers in BAL expressed in relation to total protein were more effective than if expressed in mL of BAL fluid collected.     

Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts

Microbes of the human respiratory tract are important in health and disease, but accurate sampling of the lung presents challenges. Lung microbes are commonly sampled by bronchoscopy, but to acquire samples the bronchoscope must pass through the upper respiratory tract, which is rich in microbes. Here we present methods to identify authentic lung microbiota in bronchoalveolar lavage (BAL) fluid that contains substantial oropharyngeal admixture. We studied clinical BAL samples from six selected subjects with potential heavy lung colonization. A single sample of BAL fluid was obtained from each subject along with contemporaneous oral wash (OW) to sample the oropharynx, and then DNA was extracted from three separate aliquots of each. Bacterial 16S rDNA sequences were amplified and products analyzed by 454 pyrosequencing. By comparing replicates, we were able to specify the depth of sequencing needed to reach a 95% chance of identifying a bacterial lineage of a given proportion-for example, at a depth of 5,000 tags, OTUs of proportion 0.3% or greater would be called with 95% confidence. We next constructed a single-sided outlier test that allowed lung-enriched organisms to be quantified against a background of oropharyngeal admixture, and assessed improvements available with replicate sequence analysis. This allowed identification of lineages enriched in lung in some BAL specimens. Finally, using samples from healthy volunteers collected at multiple sites in the upper respiratory tract, we show that OW provides a reasonable but not perfect surrogate for bacteria carried into to the lung by a bronchoscope. These methods allow identification of microbes that can replicate in the lung despite the background due to oropharyngeal microbes derived from aspiration and bronchoscopic carry-over.     

Bronchoalveolar lavage in interstitial lung disease: effect of volume of fluid infused

To evaluate the effect of varying infusate volume on the results of bronchoalveolar lavage (BAL) in patients with interstitial lung disease, 55 patients underwent 58 BAL during which both a 100- and 250-ml lavage was performed in the same lobe of the lung. Although the percent of the fluid that was returned and the total numbers of cells were greater in the 250- vs. the 100-ml lavage, there were no significant differences in cell differentials or numbers of cells per milliliter between the 100- and 250-ml BAL. We conclude that infusate volume does not affect cell differentials or numbers of cells per milliliter of bronchoalveolar lavage fluid in patients with interstitial lung disease.     

Chemokine Localization in Bronchial Angiogenesis

Angiogenesis in the lung involves the systemic bronchial vasculature and becomes prominent when chronic inflammation prevails. Mechanisms for neovascularization following pulmonary ischemia include growth factor transit from ischemic parenchyma to upstream bronchial arteries, inflammatory cell migration/recruitment through the perfusing artery, and paracrine effects of lung cells within the left bronchus, the niche where arteriogenesis takes place. We analyzed left lung bronchoalveolar lavage (BAL) fluid and left bronchus homogenates after left pulmonary artery ligation (LPAL) in rats, immediately after the onset of ischemia (0 h), 6 h and 24 h later. Additionally, we tested the effectiveness of dexamethasone on decreasing inflammation (0–24 h LPAL) and angiogenesis at early (3 d LPAL; bronchial endothelial proliferation) and late (14 d LPAL; blood flow) stages. After LPAL (6 h), BAL protein, total inflammatory cells, macrophages, and polymorphonuclear cells increased significantly. In parallel, pro-angiogenic CXC chemokines increased in BAL and the left main-stem bronchus (CXCL1) or only within the bronchus (CXCL2). Dexamethasone treatment reduced total BAL protein, inflammatory cells (total and polymorphonuclear cells), and CXCL1 but not CXCL2 in BAL. By contrast, no decrease was seen in either chemokine within the bronchial tissue, in proliferating bronchial endothelial cells, or in systemic perfusion of the left lung. Our results confirm the presence of CXC chemokines within BAL fluid as well as within the left mainstem bronchus. Despite significant reduction in lung injury and inflammation with dexamethasone treatment, chemokine expression within the bronchial tissue as well as angiogenesis were not affected. Our results suggest that early changes within the bronchial niche contribute to subsequent neovascularization during pulmonary ischemia.     

Serial segmental bronchoalveolar lavage in individual rats.

Bronchoalveolar lavage (BAL) is a well-characterized technique for analysis of cellular constituents of the airways and air spaces, but whole lung lavage requires that the animal be euthanized. We describe a technique of segmental BAL in rats that allows serial measurements of inflammation. A tracheal tube was placed, under direct visualization, in lightly anesthetized animals, and a catheter was passed through the tracheal tube and advanced to a wedge position. Five 0.1-ml volumes of buffer solution were instilled and then withdrawn with gentle suction. In normal rats, the percentages of neutrophils, eosinophils, and mononuclear cells had a high level of agreement in the segmental samples compared with those obtained subsequently by whole lung lavage. In rats with acute pulmonary inflammation, the differential leukocyte counts from segmental samples exhibited patterns of change that differed from those of whole lung lavage; however, most segmental samples were obtained from the left lung base so that regional variability could be minimized in serial studies. Lung mechanics and airway inflammation were not affected by repeated segmental BALs done 2 wk apart.     

Detection of lipid peroxidation in lung and in bronchoalveolar lavage cells and fluid

Inhalation of toxic materials such as asbestos, silica, 100% oxygen, ozone, or nitrogen dioxide may lead to an increased production of reactive oxygen metabolites which may initiate lipid peroxidation. Measurement of lipid peroxidation in cells and fluid obtained by bronchoalveolar lavage (BAL), as well as in lung tissue, may aid in monitoring the development and extent of pulmonary damage after inhalation of a toxic substance. In this study, we employed a sensitive assay for detection of malondialdehyde (MDA), a breakdown product of lipid peroxidation. By separation of the adduct with thiobarbituric acid, using a reverse phase high pressure liquid chromatographic technique, we accurately and sensitively measured the content of MDA in BAL cells, lavage fluid, and lavaged lung tissue homogenates of rats. The amounts of sample required for detection of MDA were small enough possibly to be applied to use with human specimens; in addition, recovery of added MDA was acceptable with all types of samples. Inclusion of a metal chelator in the preparation of samples appeared necessary to prevent metal-catalyzed propagation of lipid peroxidation during the assay. Overall, the method described here using samples from rats may be applicable to detecting lipid peroxidation in BAL samples from humans.     

Bronchoalveolar lavage fluid differential cell count. How many cells should be counted?

OBJECTIVE: To investigate the number of cells to be counted in cytocentrifuged bronchoalveolar lavage (BAL) fluid preparations in order to reach a reliable enumeration of each cell type. STUDY DESIGN: A total of 136 BAL fluid samples for patients with suspected pneumonia or interstitial lung disease were investigated. Differential cell counts were performed on May-Grünwald-Giemsa-stained cytocentrifuged preparations by 2 observers, each differentiating 500 cells. Reliability for the enumeration of each cell type was expressed as phi value, as calculated in generalizability theory. RESULTS: For polymorphonuclear neutrophils (PMNs), alveolar macrophages, lymphocytes and eosinophils, an acceptable phi value of > or = .95 was reached at a count of 300 cells by 1 observer. Mast cells reached a phi value of only .674 at a count of 500 cells by 1 observer, precluding a reliable count. At a count of 500 cells by 1 observer, squamous epithelial cells, bronchial epithelial cells and plasma cells displayed phi values of .868, .903 and .816, respectively. CONCLUSION: At a count of 300 cells, PMNs, alveolar macrophages, lymphocytes and eosinophils are reliably enumerated in cytocentrifuged BAL fluid samples.     

Immunoglobulin free light chains are increased in hypersensitivity pneumonitis and idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF), a devastating lung disorder of unknown aetiology, and chronic hypersensitivity pneumonitis (HP), a disease provoked by an immunopathologic reaction to inhaled antigens, are two common interstitial lung diseases with uncertain pathogenic mechanisms. Previously, we have shown in other upper and lower airway diseases that immunoglobulin free light chains (FLCs) are increased and may be involved in initiating a local inflammation. In this study we explored if such a mechanism may also apply to HP and IPF.

Methods

In this study we examined the presence of FLC in serum and BAL fluid from 21 IPF and 22 HP patients and controls. IgG, IgE and tryptase concentrations were measured in BAL fluid only. The presence of FLCs, plasma cells, B cells and mast cells in lung tissue of 3 HP and 3 IPF patients and 1 control was analyzed using immunohistochemistry.

Results

FLC concentrations in serum and BAL fluid were increased in IPF and HP patients as compared to control subjects. IgG concentrations were only increased in HP patients, whereas IgE concentrations were comparable to controls in both patient groups. FLC-positive cells, B cells, plasma cells, and large numbers of activated mast cells were all detected in the lungs of HP and IPF patients, not in control lung.

Conclusion

These results show that FLC concentrations are increased in serum and BAL fluid of IPF and HP patients and that FLCs are present within affected lung tissue. This suggests that FLCs may be involved in mediating pathology in both diseases.     

Leukocyte immunophenotypes in bronchoalveolar lavage fluid and peripheral blood of paracoccidioidomycosis, sarcoidosis and silicosis.

Leukocyte subsets in bronchoalveolar lavage (BAL) fluid and peripheral blood of patients with paraccoccidioidomycosis, sarcoidosis and silicosis were characterized using monoclonal antibodies and an immunoperoxidase technique. In paraccocidioidomycosis, the number of T-helper/inducer CD4-positive lymphocytes was lower in peripheral blood than in BAL fluid. Additional analysis showed that the expression of HLA-DR was very similar in alveolar macrophages, lung and blood T-cells. In sarcoidosis and silicosis there were higher proportions of T-helper/inducer cells in peripheral blood than in BAL fluid. The alterations in the T-helper/inducer/T-suppressor/cytoxic CD4/CD8 ratio in sarcoidosis and silicosis were more appreciable in peripheral blood than in BAL fluid, contrasting with the results in paracoccidioidomycosis. The expression of HLA-DR by alveolar macrophages in sarcoidosis was the highest of all the disease studied. No statistically significant differences were observed between chronic multifocal and chronic unifocal paracoccidioidomycosis disease, stage II and stage III sarcoidosis, and chronic and accelerated silicosis. The three granulomatous diseases analyzed had a few alveolar macrophages expressing the CD4 molecule on their surface. These findings and the technique of analyzing both peripheral blood and BAL leukocyte subsets may help to understand the pathogenesis of interstitial lung diseases.     

Enhanced lipid peroxidation in lung lavage of rats after inhalation of asbestos.

Exposure of phagocytic cells to asbestos in vitro results in an augmented production of reactive oxygen metabolites and increased peroxidation of lipids. The aim of this investigation was to assess the extent of lipid peroxidation both in cells and fluid obtained from bronchoalveolar lavage (BAL), and in lungs of rats exposed to crocidolite asbestos or titanium dioxide (TiO2), a nonfibrous particulate control. In comparison to sham and TiO2-exposed rats, the BAL fluid and cells of crocidolite-exposed animals contained significantly elevated levels of malondialdehyde (MDA), a breakdown product of lipid peroxidation detected using high-pressure liquid chromatography (HPLC). In contrast, no significant differences in MDA were detected in lavaged lung tissue from these animals. Inhalation of crocidolite caused an early inflammatory response characterized by elevated numbers of polymorphonuclear leukocytes and lymphocytes, as well as enhanced total protein in BAL. Pulmonary fibrosis and increased lung hydroxyproline also were observed after 20 days of exposure. Exposure to TiO2 did not cause inflammation, pulmonary fibrosis, or elevated amounts of hydroxyproline in the lung. Our results show that exposure to the fibrogenic and inflammatory mineral, crocidolite, results in an enhanced lipid peroxidation in BAL cells and fluid not observed after inhalation of the particulate TiO2. These novel observations suggest that MDA in BAL may be useful as a biomarker of exposure to inhaled asbestos or other oxidants.     

The lung at high altitude: bronchoalveolar lavage in acute mountain sickness and pulmonary edema

High-altitude pulmonary edema (HAPE), a severe form of altitude illness that can occur in young healthy individuals, is a noncardiogenic form of edema that is associated with high concentrations of proteins and cells in bronchoalveolar lavage (BAL) fluid (Schoene et al., J. Am. Med. Assoc. 256: 63-69, 1986). We hypothesized that acute mountain sickness (AMS) in which gas exchange is impaired to a milder degree is a precursor to HAPE. We therefore performed BAL with 0.89% NaCl by fiberoptic bronchoscopy in eight subjects at 4,400 m (barometric pressure = 440 Torr) on Mt. McKinley to evaluate the cellular and biochemical responses of the lung at high altitude. The subjects included one healthy control (arterial O2 saturation = 83%), three climbers with HAPE (mean arterial O2 saturation = 55.0 +/- 5.0%), and four with AMS (arterial O2 saturation = 70.0 +/- 2.4%). Cell counts and differentials were done immediately on the BAL fluid, and the remainder was frozen for protein and biochemical analysis to be performed later. The results of this and of the earlier study mentioned above showed that the total leukocyte count (X10(5)/ml) in BAL fluid was 3.5 +/- 2.0 for HAPE, 0.9 +/- 4.0 for AMS, and 0.7 +/- 0.6 for controls, with predominantly alveolar macrophages in HAPE. The total protein concentration (mg/dl) was 616.0 +/- 3.3 for HAPE, 10.4 +/- 8.3 for AMS, and 12.0 +/- 3.4 for controls, with both large- (immunoglobulin M) and small- (albumin) molecular-weight proteins present in HAPE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Shedding of soluble ICAM-1 into the alveolar space in murine models of acute lung injury

Intercellular adhesion molecule-1 (ICAM-1; CD54) is an adhesion molecule constitutively expressed in abundance on the cell surface of type I alveolar epithelial cells (AEC) in the normal lung and is a critical participant in pulmonary innate immunity. At many sites, ICAM-1 is shed from the cell surface as a soluble molecule (sICAM-1). Limited information is available regarding the presence, source, or significance of sICAM-1 in the alveolar lining fluid of normal or injured lungs. We found sICAM-1 in the bronchoalveolar lavage (BAL) fluid of normal mice (386 +/- 50 ng/ml). Additionally, sICAM-1 was spontaneously released by murine AEC in primary culture as type II cells spread and assumed characteristics of type I cells. Shedding of sICAM-1 increased significantly at later points in culture (5-7 days) compared with earlier time points (3-5 days). In contrast, treatment of AEC with inflammatory cytokines had limited effect on sICAM-1 shedding. BAL sICAM-1 was evaluated in in vivo models of acute lung injury. In hyperoxic lung injury, a reversible process with a major component of leak across the alveolar wall, BAL fluid sICAM-1 only increased in parallel with increased alveolar protein. However, in lung injury due to FITC, there were increased levels of sICAM-1 in BAL that were independent of changes in BAL total protein concentration. We speculate that after lung injury, changes in sICAM-1 in BAL fluid are associated with progressive injury and may be a reflection of type I cell differentiation during reepithelialization of the injured lung.     

Expression of transforming growth factor beta (TGF-b1) by human preterm lung inflammatory cells

Using a previously published model of human BPD this study examines whether preterm lung inflammatory cells produce transforming growth factor beta 1 (TGF-beta1), a cytokine pivotal in pathogenesis of bronchopulmonary dysplasia (BPD), and whether TGF-beta1 expression is regulated by inflammation. Lung inflammatory cells (neutrophils and macrophages) recovered in the broncho-alveolar (BAL) fluid of premature infants intubated for respiratory distress after birth expressed TGF-b1 mRNA and protein. Total and bioactive TGF-beta1 were abundantly found in the BAL fluid of the same infants. In cell culture stimulation by lipopolysaccharide (LPS) did not result in any further expression of total or bioactive TGF-beta1 by neonatal lung inflammatory cells over constitutive concentrations. In conclusion, lung inflammatory cells from premature infants are a source of TGF-beta1 but LPS does not regulate TGF-b1 production in these cells.     

Alveolar macrophages contribute to alveolar barrier dysfunction in ventilator-induced lung injury

In patients requiring mechanical ventilation for acute lung injury or acute respiratory distress syndrome (ARDS), tidal volume reduction decreases mortality, but the mechanisms of the protective effect have not been fully explored. To test the hypothesis that alveolar macrophage activation is an early and critical event in the initiation of ventilator-induced lung injury (VILI), rats were ventilated with high tidal volume (HV(T)) for 10 min to 4 h. Alveolar macrophage counts in bronchoalveolar lavage (BAL) fluid decreased 45% by 20 min of HV(T) (P < 0.05) consistent with activation-associated adhesion. Depletion of alveolar macrophages in vivo with liposomal clodronate significantly decreased permeability and pulmonary edema following 4 h of HV(T) (P < 0.05). BAL fluid from rats exposed to 20 min of HV(T) increased nitric oxide synthase activity nearly threefold in na?ve primary alveolar macrophages (P < 0.05) indicating that soluble factors present in the air spaces contribute to macrophage activation in VILI. Media from cocultures of alveolar epithelial cell monolayers and alveolar macrophages exposed to 30 min of stretch in vitro also significantly increased nitrite production in na?ve macrophages (P < 0.05), but media from stretched alveolar epithelial cells or primary alveolar macrophages alone did not, suggesting alveolar epithelial cell-macrophage interaction was required for the subsequent macrophage activation observed. These data demonstrate that injurious mechanical ventilation rapidly activates alveolar macrophages and that alveolar macrophages play an important role in the initial pathogenesis of VILI.     

What is the clinical relevance of different lung compartments?

The lung consists of at least seven compartments with relevance to immune reactions. Compartment 1 - the bronchoalveolar lavage (BAL), which represents the cells of the bronchoalveolar space: From a diagnostic point of view the bronchoalveolar space is the most important because it is easily accessible in laboratory animals, as well as in patients, using BAL. Although this technique has been used for several decades it is still unclear to what extent the BAL represents changes in other lung compartments. Compartment 2 - bronchus-associated lymphoid tissue (BALT): In the healthy, BALT can be found only in childhood. The role of BALT in the development of the mucosal immunity of the pulmonary surfaces has not yet been resolved. However, it might be an important tool for inhalative vaccination strategies. Compartment 3 - conducting airway mucosa: A third compartment is the bronchial epithelium and the submucosa, which both contain a distinct pool of leukocytes (e.g. intraepithelial lymphocytes, IEL). This again is also accessible via bronchoscopy. Compartment 4 - draining lymph nodes/Compartment 5 - lung parenchyma: Transbronchial biopsies are more difficult to perform but provide access to two additional compartments - lymph nodes with the draining lymphatics and lung parenchyma, which roughly means "interstitial" lung tissue. Compartment 6 - the intravascular leukocyte pool: The intravascular compartment lies between the systemic circulation and inflamed lung compartments. Compartment 7 - periarterial space: Finally, there is a unique, lung-specific space around the pulmonary arteries which contains blood and lymph capillaries. There are indications that this "periarterial space" may be involved in the pulmonary host defense. All these compartments are connected but the functional network is not yet fully understood. A better knowledge of the complex interactions could improve diagnosis and therapy, or enable preventive approaches of local immunization.     

The role of transbronchial lung biopsy in the diagnosis of solitary pulmonary nodule

Transbronchial lung biopsy (TBLB) is a well-recognized diagnostic technique in diffuse interstitial lung diseases, but it is not considered to be the first choice in investigation of solitary pulmonary nodules (SPN). The main idea of this study was to increase the sensitivity of bronchoscopy using multiple techniques, especially TBLB, thus to avoid more aggressive diagnostic procedures. The objective of this prospective study was to evaluate the efficacy and safety of TBLB in the diagnosis of SPN, in comparison with other bronchoscopic techniques. Fifty patients with chest x-ray finding consistent with SPN underwent bronchoscopy with bronchial washing, brushing, bronchoalveolar lavage (BAL) and TBLB were included in this study. Thirty-one patients suffered from malignant tumors, while 19 patients had nonmalignant lesions. TBLB achieved overall diagnostic sensitivity of 62%, BAL of 29%, bronchial brushing of 16% and washing of 6%. Combining all techniques together, bronchoscopy had overall sensitivity of 86%. Concerning malignant lesions, TBLB had a sensitivity of 65%, specificity of 100%, and accuracy of 82%. TBLB had a significantly better yield for lesions with a diameter > or = 25 mm than for lesions of < 25 mm (sensitivity of 82% and 53% respectively, p < 0.05). Diagnostic yield improved significantly with the increasing number of specimens (less than 3 specimens: sensitivity 59%, 3 or more specimens: sensitivity 87%, p < 0.05). Complications of TBLB occurred in 2 (4%) patients: 1 incomplete pneumothorax and 1 hemorrhage. According to the results, we conclude that TBLB is an accurate and safe technique for the diagnosis of pulmonary solitary nodule with a diameter equal or greater than 25 mm.     

Essential contribution of monocyte chemoattractant protein-1/C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia

Neutrophil infiltration is the first step in eradication of bacterial infection, but neutrophils rapidly die after killing bacteria. Subsequent accumulation of macrophage lineage cells, such as alveolar macrophages (AMs), is essential to remove dying neutrophils, which are a source of injurious substances. Macrophage lineage cells can promote tissue repair, by producing potential growth factors including hepatocyte growth factor (HGF). However, it remains elusive which factor activates macrophage in these processes. Intratracheal instillation of Pseudomonas aeruginosa caused neutrophil infiltration in the airspace; subsequently, the numbers of total AMs and neutrophil ingested AMs were increased. Bronchoalveolar lavage (BAL) fluid levels of monocyte chemoattractant protein (MCP)-1/CC chemokine ligand-2 (CCL2), a potent macrophage-activating factor, were increased before the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid. Immunoreactive MCP-1 proteins were detected in alveolar type II epithelial cells and AMs only after P. aeruginosa infection. The administration of anti-MCP-1/CCL2 Abs reduced the increases in the number of AM-ingesting neutrophils and HGF levels in BAL fluid, and eventually aggravated lung tissue injury. In contrast, the administration of MCP-1/CCL2 enhanced the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid, and eventually attenuated lung tissue injury. Furthermore, MCP-1/CCL2 enhanced the ingestion of apoptotic neutrophils and HGF production by a mouse macrophage cell line, RAW 267.4, in a dose-dependent manner. Collectively, MCP-1/CCL2 has a crucial role in the resolution and repair processes of acute bacterial pneumonia by enhancing the removal of dying neutrophils and HGF production by AMs.