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1.  Intrapulmonary Distribution and Pharmacokinetics of Laninamivir, a Neuraminidase Inhibitor, after a Single Inhaled Administration of Its Prodrug, Laninamivir Octanoate, in Healthy Volunteers 
A single inhaled dose of laninamivir octanoate (LO), a long-acting neuraminidase inhibitor, exhibits efficacy in treating both adult and pediatric patients with influenza virus infection. The intrapulmonary pharmacokinetics (PK) of LO and laninamivir, a pharmacologically active metabolite, were investigated by a single-center, open-label study of healthy adult volunteers. Subgroups of five subjects each underwent bronchoalveolar lavage (BAL) 4, 8, 24, 48, 72, 168, and 240 h following a single inhaled administration of LO (40 mg). Plasma, BAL fluid, and alveolar macrophages (AM) were analyzed to determine LO and laninamivir concentrations, using validated liquid chromatography-tandem mass spectrometry methods. The concentrations in epithelial lining fluid (ELF) and AM from the first and subsequent BAL fluid samples were determined separately to explore the drug distribution in airways. Mean laninamivir concentrations in ELF, calculated using the first BAL fluids and BAL fluids collected 4 h after inhaled administration, were 8.57 and 2.40 μg/ml, respectively. The laninamivir concentration in ELF decreased with a longer half-life than that in plasma, and it exceeded the 50% inhibitory concentrations for viral neuraminidases at all time points examined for 240 h after the inhalation. Laninamivir exposure in ELF from the first BAL samples was 3.2 times higher than that in ELF from the subsequent BAL fluid samples. ELF concentration profiles of laninamivir support its long-lasting effect for treatment of patients with influenza virus infection by a single inhaled administration.
PMCID: PMC3393442  PMID: 22526307
2.  Alveolar Macrophage Gene Expression is altered in the Setting of Alcohol Use Disorders 
Alcohol use disorders (AUDs) are associated with an increased susceptibility to a variety of common and devastating pulmonary diseases including community- and hospital-acquired pneumonias, as well as the acute respiratory distress syndrome (ARDS). Alveolar macrophages play an important role in preventing the development of these disorders through maintaining lung sterility and resolving lung inflammation. Although alcohol exposure has been associated with aberrant alveolar macrophage function in animal models, the clinical relevance of these observations in humans is not established. Therefore, we sought to determine the effects of AUDs on human alveolar macrophage gene expression.
Whole genome microarray analysis was performed on alveolar macrophages obtained by bronchoalveolar lavage from a test cohort of subjects with AUDs (n=7), and controls (n=7) who were pair-matched on age, gender, and smoking. Probe set expression differences in this cohort were validated by real time reverse transcription-polymerase chain reaction (RT RT-PCR). Functional analysis with web-based bioinformatics tools was utilized with microarray data to assess differentially expressed candidate genes (p<0.01) based on alcohol consumption. Alveolar macrophage mRNA samples from a second cohort of subjects with AUDs (n=7) and controls (n=7) were used to confirm gene expression differences related to AUDs. Results: In both the test and confirmatory cohorts, AUDs were associated with upregulation of alveolar macrophage gene expression related to apoptosis, including perforin-1, granzyme A, and CXCR4 (fusin). Pathways governing the regulation of progression through cell cycle and immune response were also affected, as was upregulation of gene expression for mitochondrial superoxide dismutase. Overall, 12 genes’ expression was affected by AUDs independent of smoking.
AUDs are associated with unique changes in human alveolar macrophage gene expression. Novel therapies targeting alveolar macrophage gene expression in the setting of AUDs may prove to be clinically useful in limiting susceptibility for pulmonary disorders in these individuals.
PMCID: PMC3059108  PMID: 21121937
pneumonia; acute lung injury; apoptosis; perforin; human
3.  Relationship between levels of secreted phospholipase A2 groups IIA and X in the airways and asthma severity 
Secreted phospholipase A2s (sPLA2) may be important mediators of asthma, but the specific sPLA2s involved in asthma are not known.
To evaluate sPLA2 group IIA, V, and X proteins (sPLA2-IIA, sPLA2-V and sPLA2-X) in bronchoalveolar lavage (BAL) fluid, BAL cells and airway epithelial cells of subjects with and without asthma, and examine the relationship between the levels of specific sPLA2 enzymes and airway inflammation, asthma severity, and lung function.
The expression of sPLA2-IIA, sPLA2-V and sPLA2-X in BAL cells and epithelial brushings was assessed by qPCR. The levels of these sPLA2 proteins and sPLA2 activity with and without group II and group X-specific inhibitors were measured in BAL fluid from 18 controls and 39 asthmatics.
The airway epithelium expressed sPLA2-X at higher levels than either sPLA2-IIA or sPLA2-V, whereas BAL cells expressed sPLA2-IIA and sPLA2-X at similar levels. The majority of sPLA2 activity in BAL fluid was attributed to either sPLA2-IIA or sPLA2-X. After 10-fold concentration of BAL fluid, the levels of sPLA2-X normalized to total protein were increased in asthma and were associated with lung function, the concentration of induced sputum neutrophils, and prostaglandin E2. The levels of sPLA2-IIA were elevated in asthma when normalized to total protein, but where not related to lung function, markers of airway inflammation or eicosanoid formation.
Conclusions and Clinical Relevance
These data indicate that sPLA2-IIA and sPLA2-X are the major sPLA2s in human airways, and suggest a link between the levels of sPLA2-X in the airways and several features of asthma.
PMCID: PMC3093436  PMID: 21255140
Asthma; Eicosanoid; Epithelial Cell; Leukotriene; Phospholipase
4.  Interpretation of Epithelial Lining Fluid Concentrations of Antibiotics against Methicillin Resistant Staphylococcus aureus 
Infection & Chemotherapy  2014;46(4):219-225.
Although antibiotics whose epithelial lining fluid (ELF) concentrations are reported high tend to be preferred in treatment of pneumonia, measurement of ELF concentrations of antibiotics could be misled by contamination from lysis of ELF cells and technical errors of bronchoalveolar lavage (BAL). In this review, ELF concentrations of anti-methicillin resistant Staphylococcus aureus (MRSA) antibiotics were interpreted considering above confounding factors. An equation used to explain antibiotic diffusion into CSF (cerebrospinal fluid) was adopted: ELF/free serum concentration ratio = 0.96 + 0.091 × ln (partition coefficient / molecular weight1/2). Seven anti-MRSA antibiotics with reported ELF concentrations were fitted to this equation to see if their ELF concentrations were explainable by the penetration capacity only. Then, outliers were modeled under the assumption of varying contamination from lysed ELF cells (test range 0-10% of ELF volume). ELF concentrations of oritavancin, telavancin, tigecycline, and vancomycin were well described by the diffusion equation, with or without additional impact from cell lysis. For modestly high ELF/free serum concentration ratio of linezolid, technical errors of BAL should be excluded. Although teicoplanin and iclaprim showed high ELF/free serum ratios also, their protein binding levels need to be cleared for proper interpretation. At the moment, it appears very premature to use ELF concentrations of anti-MRSA antibiotics as a relevant guide for treatment of lung infections by MRSA.
PMCID: PMC4285007  PMID: 25566401
Epithelial lining fluid; Methicillin-resistant Staphylococcus aureus; Bronchoalveolar lavage; Protein binding
5.  Increased concentrations of human ß-defensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis 
Thorax  2003;58(5):425-430.
Methods: The concentrations of HBD-1 and HBD-2 in plasma and bronchoalveolar lavage (BAL) fluid from 33 patients with DPB and 30 normal adults were measured by radioimmunoassay. Localisation of HBD-2 was investigated immunohistochemically in an open lung biopsy specimen obtained from a patient with DPB.
Results: High concentrations of HBD-1 and HBD-2 were noted in BAL fluid from DPB patients. Increased plasma concentrations of HBD-2, but not HBD-1, were found in patients with DPB compared with control subjects. In patients with DPB the HBD-2 concentration in BAL fluid correlated significantly with the numbers of cells recovered from the BAL fluid (total cells, neutrophils, and lymphocytes) and with the BAL fluid concentration of IL-1ß. Synthetic HBD-2, but not HBD-1, had dose dependent bactericidal activity against P aeruginosa. Treatment of 14 patients with macrolides significantly reduced BAL fluid concentrations of HBD-2 but not HBD-1 or plasma concentrations of HBD-1 and HBD-2. Immunohistochemistry of lung tissue showed localisation of HBD-2 in the epithelia of the distal bronchioles.
Conclusions: These results indicate that ß-defensins, particularly HBD-2, participate in antimicrobial defence in the respiratory tract in DPB, and that the BAL fluid concentration of HBD-2 may be a useful marker of airway inflammation in patients with DPB.
PMCID: PMC1746672  PMID: 12728165
6.  Intrapulmonary Pharmacokinetics of Linezolid 
In this study, our objective was to determine the steady-state intrapulmonary concentrations and pharmacokinetic parameters of orally administered linezolid in healthy volunteers. Linezolid (600 mg every 12 h for a total of five doses) was administered orally to 25 healthy adult male subjects. Each subgroup contained five subjects, who underwent bronchoscopy and bronchoalveolar lavage (BAL) 4, 8, 12, 24, or 48 h after administration of the last dose. Blood was obtained for drug assay prior to administration of the first dose and fifth dose and at the completion of bronchoscopy and BAL. Standardized bronchoscopy was performed without systemic sedation. The volume of epithelial lining fluid (ELF) recovered was calculated by the urea dilution method, and the total number of alveolar cells (AC) was counted in a hemocytometer after cytocentrifugation. Linezolid was measured in plasma by a high-pressure liquid chromatography (HPLC) technique and in BAL specimens and AC by a combined HPLC-mass spectrometry technique. Areas under the concentration-time curves (AUCs) for linezolid in plasma, ELF, and AC were derived by noncompartmental analysis. Half-lives for linezolid in plasma, ELF, and AC were calculated from the elimination rate constants derived from a monoexponential fit of the means of the observed concentrations at each time point. Concentrations (means ± standard deviations) in plasma, ELF, and AC, respectively, were 7.3 ± 4.9, 64.3 ± 33.1, and 2.2 ± 0.6 μg/ml at the 4-h BAL time point and 7.6 ± 1.7, 24.3 ± 13.3, and 1.4 ± 1.3 μg/ml at the 12-h BAL time point. Linezolid concentrations in plasma, ELF, and AC declined monoexponentially, with half-lives of 6.9, 7.0, and 5.7 h, respectively. For a MIC of 4, the 12-h plasma AUC/MIC and maximum concentration/MIC ratios were 34.6 and 3.9, respectively, and the percentage of time the drug remained above the MIC for the 12-h dosing interval was 100%; the corresponding ratios in ELF were 120 and 16.1, respectively, and the percentage of time the drug remained above the MIC was 100%. The long plasma and intrapulmonary linezolid half-lives and the percentage of time spent above the MIC of 100% of the dosing interval provide a pharmacokinetic rationale for drug administration every 12 h and indicate that linezolid is likely to be an effective agent for the treatment of pulmonary infections.
PMCID: PMC127139  PMID: 11959585
7.  Usefulness of Cellular Analysis of Bronchoalveolar Lavage Fluid for Predicting the Etiology of Pneumonia in Critically Ill Patients 
PLoS ONE  2014;9(5):e97346.
The usefulness of bronchoalveolar lavage (BAL) fluid cellular analysis in pneumonia has not been adequately evaluated. This study investigated the ability of cellular analysis of BAL fluid to differentially diagnose bacterial pneumonia from viral pneumonia in adult patients who are admitted to intensive care unit.
BAL fluid cellular analysis was evaluated in 47 adult patients who underwent bronchoscopic BAL following less than 24 hours of antimicrobial agent exposure. The abilities of BAL fluid total white blood cell (WBC) counts and differential cell counts to differentiate between bacterial and viral pneumonia were evaluated using receiver operating characteristic (ROC) curve analysis.
Bacterial pneumonia (n = 24) and viral pneumonia (n = 23) were frequently associated with neutrophilic pleocytosis in BAL fluid. BAL fluid median total WBC count (2,815/µL vs. 300/µL, P<0.001) and percentage of neutrophils (80.5% vs. 54.0%, P = 0.02) were significantly higher in the bacterial pneumonia group than in the viral pneumonia group. In ROC curve analysis, BAL fluid total WBC count showed the best discrimination, with an area under the curve of 0.855 (95% CI, 0.750–0.960). BAL fluid total WBC count ≥510/µL had a sensitivity of 83.3%, specificity of 78.3%, positive likelihood ratio (PLR) of 3.83, and negative likelihood ratio (NLR) of 0.21. When analyzed in combination with serum procalcitonin or C-reactive protein, sensitivity was 95.8%, specificity was 95.7%, PLR was 8.63, and NLR was 0.07. BAL fluid total WBC count ≥510/µL was an independent predictor of bacterial pneumonia with an adjusted odds ratio of 13.5 in multiple logistic regression analysis.
Cellular analysis of BAL fluid can aid early differential diagnosis of bacterial pneumonia from viral pneumonia in critically ill patients.
PMCID: PMC4019586  PMID: 24824328
8.  Free radical activity and pro-inflammatory effects of particulate air pollution (PM10) in vivo and in vitro. 
Thorax  1996;51(12):1216-1222.
BACKGROUND: Epidemiological evidence has implicated fine particulate air pollution, particularly particles less than 10 microns in diameter (PM10), in the development of exacerbations of asthma and chronic obstructive pulmonary disease (COPD) although the mechanism is unknown. The hypothesis that PM10 particles induce oxidant stress, causing inflammation and injury to airway epithelium, was tested. METHODS: The effects of intratracheal instillation of PM10 was assessed in rat lungs (three per group). Inflammatory cell influx was measured by bronchoalveolar lavage (BAL) and air space epithelial permeability was assessed as the total protein in BAL fluid in vivo. The oxidant properties of PM10 particles were determined by their ability to cause damage to plasmid DNA and by changes in reduced (GSH) and oxidised (GSSG) glutathione. The effects of PM10 particles were compared in some experiments with those of fine (CB) and ultrafine (ufCB) carbon black particles. RESULTS: Six hours after intratracheal instillation of PM10 there was an influx of neutrophils (up to 15% of total cells in BAL fluid) into the alveolar space, increased epithelial permeability, the mean (SE) total protein in the BAL fluid increasing from 0.39 (0.01) to 0.62 (0.01) mg/ml, and increased lactate dehydrogenase (LDH) concentrations in the BAL fluid. An even greater inflammatory response was seen following intratracheal instillation of ufCB but not following CB instillation. PM10 particles had free radical activity in vivo, as shown by a decrease in GSH levels in the BAL fluid from 0.36 (0.05) to 0.25 (0.01) nmol/ml following instillation. The free radical activity of PM10 was confirmed in vitro by its ability to deplete supercoiled plasmid DNA, an effect which could be reversed by mannitol, a specific hydroxyl radical scavenger. BAL fluid leucocytes from rats treated with PM10 produced greater amounts of nitric oxide (NO), measured as nitrite (control 3.07 (0.33), treated 4.45 (0.23) microM/1 x 10(6) cells), and tumour necrosis factor alpha (control 21.0 (3.1), treated 179.2 (29.4) units/l x 10(6) cells) in culture than those obtained from control animals. Since the PM10 preparation was contaminated with small amounts of filter fibres due to the extraction process, the effects of instillation of filter fibres alone was assessed. These studies showed that filter fibres did not account for the proinflammatory and injurious effects of the PM10 suspension. CONCLUSIONS: These findings provide evidence that PM10 has free radical activity and causes lung inflammation and epithelial injury. These data support the proposed hypothesis for the mechanism by which particulate air pollution causes adverse effects in patients with airways diseases.
PMCID: PMC472766  PMID: 8994518
9.  A role for CFTR in the elevation of glutathione levels in the lung by oral glutathione administration 
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is the only known apical glutathione (GSH) transporter in the lung. The purpose of these studies was to determine whether oral GSH or glutathione disulfide (GSSG) treatment could increase lung epithelial lining fluid (ELF) GSH levels and whether CFTR plays a role in this process. The pharmacokinetic profile of an oral bolus dose of GSH (300 mg/kg) was determined in mice. Plasma, ELF, bronchoalveolar lavage (BAL) cells, and lung tissue were analyzed for GSH content. There was a rapid elevation in the GSH levels that peaked at 30 min in the plasma and 60 min in the lung, ELF, and BAL cells after oral GSH dosing. Oral GSH treatment produced a selective increase in the reduced and active form of GSH in all lung compartments examined. Oral GSSG treatment (300 mg/kg) resulted in a smaller increase of GSH levels. To evaluate the role of CFTR in this process, Cftr knockout (KO) mice and gut-corrected Cftr KO-transgenic (Tg) mice were given an oral bolus dose of GSH (300 mg/kg) and compared with wild-type mice for changes in GSH levels in plasma, lung, ELF, and BAL cells. There was a twofold increase in plasma, a twofold increase in lung, a fivefold increase in ELF, and a threefold increase in BAL cell GSH levels at 60 min in wild-type mice; however, GSH levels only increased by 40% in the plasma, 60% in the lung, 50% in the ELF, and twofold in the BAL cells within the gut-corrected Cftr KO-Tg mice. No change in GSH levels was observed in the uncorrected Cftr KO mice. These studies suggest that CFTR plays an important role in GSH uptake from the diet and transport processes in the lung.
PMCID: PMC3983954  PMID: 17369290
nutrition; transport; high-performance liquid chromatography; mice; cystic fibrosis; antioxidant; cystic fibrosis transmembrane conductance regulator
10.  Quantification of Amikacin in Bronchial Epithelial Lining Fluid in Neonates▿ 
Amikacin efficacy is based on peak concentrations and the possibility of reaching therapeutic levels at the infection site. This study aimed to describe amikacin concentrations in the epithelial lining fluid (ELF) through bronchoalveolar lavage (BAL) in newborns. BAL fluid was collected in ventilated neonates treated with intravenous (i.v.) amikacin. Clinical characteristics, amikacin therapeutic drug monitoring serum concentrations, and the concentrations of urea in plasma were extracted from the individual patient files. Amikacin and urea BAL fluid concentrations were determined using liquid chromatography with pulsed electrochemical detection (LC-PED) and capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D), respectively. ELF amikacin concentrations were converted from BAL fluid concentrations through quantification of dilution (urea in plasma/urea in BAL fluid) during the BAL procedure. Twenty-two observations in 17 neonates (postmenstrual age, 31.9 [range, 25.1 to 41] weeks; postnatal age, 3.5 [range, 2 to 37] days) were collected. Median trough and peak amikacin serum concentrations were 2.1 (range, 1 to 7.1) mg/liter and 39.1 (range, 24.1 to 73.2) mg/liter; the median urea plasma concentration was 30 (8 to 90) mg/dl. The median amikacin concentration in ELF was 6.5 mg/liter, the minimum measured concentration was 1.5 mg/liter, and the maximum (peak) was 23 mg/liter. The highest measured ELF concentration was reached between 6 and 14.5 h after i.v. amikacin administration, and an estimated terminal elimination half-life was 8 to 10 h. The median and highest (peak) ELF amikacin concentrations observed in our study population were, respectively, 6.5 and 23 mg/liter. Despite the frequent use of amikacin in neonatal (pulmonary) infections, this is the first report of amikacin quantification in ELF in newborns.
PMCID: PMC3165321  PMID: 21709076
11.  Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid 
The diagnosis of invasive pulmonary aspergillosis (IPA) remains challenging. Culture and histopathological examination of bronchoalveolar lavage (BAL) fluid are useful but have suboptimal sensitivity and in the case of culture may require several days for fungal growth to be evident. Detection of Aspergillus DNA in BAL fluid by quantitative PCR (qPCR) offers the potential for earlier diagnosis and higher sensitivity. It is important to adopt quality control measures in PCR assays to address false positives and negatives which can hinder accurate evaluation of diagnostic performance.
BAL fluid from 94 episodes of pneumonia in 81 patients was analyzed. Thirteen episodes were categorized as proven or probable IPA using Mycoses Study Group criteria. The pellet and the supernatant fractions of the BAL were separately assayed. A successful extraction was confirmed with a human 18S rRNA gene qPCR. Inhibition in each qPCR was measured using an exogenous DNA based internal amplification control (IAC). The presence of DNA from pathogens in the Aspergillus genus was detected using qPCR targeting fungal 18S rRNA gene.
Human 18S rRNA gene qPCR confirmed successful DNA extraction of all samples. IAC detected some degree of initial inhibition in 11 samples. When culture was used to diagnose IPA, the sensitivity and specificity were 84.5% and 100% respectively. Receiver-operating characteristic analysis of qPCR showed that a cutoff of 13 fg of Aspergillus genomic DNA generated a sensitivity, specificity, positive and negative predictive value of 77%, 88%, 50%, 96% respectively. BAL pellet and supernatant analyzed together resulted in sensitivity and specificity similar to BAL pellet alone. Some patients did not meet standard criteria for IPA, but had consistently high levels of Aspergillus DNA in BAL fluid by qPCR.
The Aspergillus qPCR assay detected Aspergillus DNA in 76.9% of subjects with proven or probable IPA when the concentrated BAL fluid pellet fraction was used for diagnosis. There was no benefit from analyzing the BAL supernatant fraction. Use of both extraction and amplification controls provided optimal quality control for interpreting qPCR results and therefore may increase our understanding of the true potential of qPCR for the diagnosis of IPA.
PMCID: PMC2440748  PMID: 18510764
12.  Bacterial Yield from Quantitative Cultures of Bronchoalveolar Lavage Fluid in Patients with Pneumonia on Antimicrobial Therapy 
Early diagnosis and appropriate antimicrobial choice are crucial when managing pneumonia patients, and quantitative culture of bronchoalveolar lavage (BAL) fluid is considered a useful method for identifying pneumonia pathogens. We evaluated the quantitative yield of BAL fluid bacterial cultures in patients being treated with antimicrobials and attempted to identify factors predictive of positive BAL cultures.
Patients over 18 years old and whose BAL fluid was subjected to quantitative culture to identify the organism causative of pneumonia between January 1, 2005, and December 31, 2009, were included. We reviewed the results of BAL fluid bacterial cultures and the clinical records, laboratory tests, and radiographic findings of the patients.
BAL was performed on 340 patients with pneumonia. A positive BAL culture, defined as isolation of more than 104 colony forming units/mL bacteria, was documented in 18 (5.29%) patients. Of these, 9 bacteria isolated from 10 patients were classified as probable pathogens. The most frequently isolated bacteria were methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. No independent predictive factor for positive BAL cultures was identified.
The yield of quantitative BAL fluid bacterial culture in patients already on antimicrobials was low. Clinicians should be cautious when performing a BAL culture in patients with pneumonia who are already on antimicrobials.
PMCID: PMC3372799  PMID: 22707887
Bronchoalveolar lavage; Pneumonia; Antimicrobials
13.  Intrapulmonary Pharmacokinetics of Telithromycin, a New Ketolide, in Healthy Japanese Volunteers 
The concentrations of telithromycin, a new ketolide antimicrobial agent, in alveolar macrophages (AMs) and bronchoalveolar epithelial lining fluid (ELF) were determined in order to investigate the transfer of the drug into target tissue, relative to plasma, following multiple oral doses of telithromycin. Twenty-four healthy male Japanese volunteers were randomly allocated to four groups. Each subject was given 600 or 800 mg of telithromycin once daily for 5 days, followed by bronchoalveolar lavage (BAL) 2 or 8 h after the last dose (group A and B: 600 mg, 2 and 8 h BAL time point; group C and D: 800 mg, 2 and 8 h BAL time point). The mean concentrations of the drug in AMs and ELF were 34.54 and 4.92 mg/liter in group A, 50.97 and 2.26 mg/liter in group B, 25.47 and 4.24 mg/liter in group C, and 108.22 and 4.31 mg/liter in group D, respectively, which markedly exceeded concentrations in plasma. These results demonstrated good transfer of telithromycin into AMs and ELF, suggesting good efficacy against common respiratory pathogens, including intracellular pathogens and atypical microorganisms.
PMCID: PMC127464  PMID: 11850288
14.  Lactobacillus casei modulates the inflammation-coagulation interaction in a pneumococcal pneumonia experimental model 
We have previously demonstrated that Lactobacillus casei CRL 431 administration improved the resistance to pneumococcal infection in a mouse model.
This study examined the effects of the oral administration of Lactobacillus casei CRL 431 (L. casei) on the activation of coagulation and fibrinolytic systems as well as their inhibitors during a Streptococcus pneumoniae infection in mice.
The alveolo-capillary membrane was damaged and the coagulation system was also activated by the infection. As a consequence, we could see fibrin(ogen) deposits in lung histological slices, increased levels of thrombin-antithrombin complex (TATc) in bronchoalveolar lavage (BAL) and plasma, decrease in prothrombin activity (PT) and prolonged activated partial thromboplastin time test (APTT) values. Factor VII (FVII) and factor X (FX) were decreased in plasma, whereas fibrinogen (F) and factor VIII (FVIII) were increased. The low levels of protein C (PC) in BAL and plasma proved damage on inhibitory activity. The infected animals showed reduced fibrinolytic activity, evidenced by an increase in plasminogen activation inhibitor-1 (PAI-1) in BAL and plasma. The pathogen induced an increase of TNF-α, IL-1β and IL-6 in BAL and serum a few hours after challenge followed by a significant decrease until the end of the assayed period. IL-4 and IL-10 in BAL and serum were also augmented, especially at the end of the experiment. The animals treated with L. casei showed an improvement of alveolo-capillary membrane, lower fibrin(ogen) deposits in lung and decrease in TATc. APTT test and PT, FVII and FX activity were normalized. L. casei group showed lower F levels than control during whole experiment. In the present study no effect of L. casei on the recovery of the inhibitory activity was detected. However, L. casei was effective in reducing PAI-1 levels in BAL and in increasing anti-inflammatory ILs concentration.
L. casei proved effective to regulate coagulation activation and fibrinolysis inhibition during infection, leading to a decrease in fibrin deposits in lung. This protective effect of L. casei would be mediated by the induction of higher levels of IL-4 and IL-10 which could regulate the anti-inflammatory, procoagulant and antifibrinolytic effects of TNF-α, IL-1β and IL-6.
PMCID: PMC2770469  PMID: 19835595
15.  Budesonide/Formoterol Enhances the Expression of Pro Surfactant Protein-B in Lungs of COPD Patients 
PLoS ONE  2013;8(12):e83881.
Rationale & Aim
Pulmonary surfactants are essential components of lung homeostasis. In chronic obstructive pulmonary disease (COPD), surfactant expression decreases in lungs whereas, there is a paradoxical increase in protein expression in plasma. The latter has been associated with poor health outcomes in COPD. The purpose of this study was to determine the relationship of surfactants and other pneumoproteins in bronchoalveolar lavage (BAL) fluid and plasma to airflow limitation and the effects of budesonide/formoterol on this relationship.
We recruited (clinical identifier: NCT00569712) 7 smokers without COPD and 30 ex and current smokers with COPD who were free of exacerbations for at least 4 weeks. All subjects were treated with budesonide/formoterol 400/12 µg twice a day for 4 weeks. BAL fluid and plasma samples were obtained at baseline and the end of the 4 weeks. We measured lung-predominant pneumoproteins: pro-Surfactant Protein-B (pro-SFTPB), Surfactant Protein-D (SP-D), Club Cell Secretory Protein-16 (CCSP-16) and Pulmonary and Activation-Regulated Chemokine (PARC/CCL-18) in BAL fluid and plasma.
BAL Pro-SFTPB concentrations had the strongest relationship with airflow limitation as measured by FEV1/FVC (Spearman rho = 0.509; p = 0.001) and FEV1% of predicted (Spearman rho =  0.362; p = 0.028). Plasma CCSP-16 concentrations were also significantly related to airflow limitation (Spearman rho = 0.362; p = 0.028 for FEV1% of predicted). The other biomarkers in BAL fluid or plasma were not significantly associated with airflow limitation. In COPD subjects, budesonide/formoterol significantly increased the BAL concentrations of pro-SFTPB by a median of 62.46 ng/ml (p = 0.022) or 48.7% from baseline median value.
Increased severity of COPD is associated with reduced Pro-SFTPB levels in BAL fluid. Short-term treatment with budesonide/formoterol increases these levels in BAL fluid. Long term studies will be needed to determine the clinical relevance of this observation.
PMCID: PMC3873417  PMID: 24386300
16.  Keratinocyte Growth Factor Promotes Epithelial Survival and Resolution in a Human Model of Lung Injury 
Rationale: Increasing epithelial repair and regeneration may hasten resolution of lung injury in patients with the acute respiratory distress syndrome (ARDS). In animal models of ARDS, keratinocyte growth factor (KGF) reduces injury and increases epithelial proliferation and repair. The effect of KGF in the human alveolus is unknown.
Objectives: To test whether KGF can attenuate alveolar injury in a human model of ARDS.
Methods: Volunteers were randomized to intravenous KGF (60 μg/kg) or placebo for 3 days, before inhaling 50 μg LPS. Six hours later, subjects underwent bronchoalveolar lavage (BAL) to quantify markers of alveolar inflammation and cell-specific injury.
Measurements and Main Results: KGF did not alter leukocyte infiltration or markers of permeability in response to LPS. KGF increased BAL concentrations of surfactant protein D, matrix metalloproteinase (MMP)-9, IL-1Ra, granulocyte-macrophage colony–stimulating factor (GM-CSF), and C-reactive protein. In vitro, BAL fluid from KGF-treated subjects inhibited pulmonary fibroblast proliferation, but increased alveolar epithelial proliferation. Active MMP-9 increased alveolar epithelial wound repair. Finally, BAL from the KGF-pretreated group enhanced macrophage phagocytic uptake of apoptotic epithelial cells and bacteria compared with BAL from the placebo-treated group. This effect was blocked by inhibiting activation of the GM-CSF receptor.
Conclusions: KGF treatment increases BAL surfactant protein D, a marker of type II alveolar epithelial cell proliferation in a human model of acute lung injury. Additionally, KGF increases alveolar concentrations of the antiinflammatory cytokine IL-1Ra, and mediators that drive epithelial repair (MMP-9) and enhance macrophage clearance of dead cells and bacteria (GM-CSF).
Clinical trial registered with ISRCTN 98813895.
PMCID: PMC4226011  PMID: 24716610
acute respiratory distress syndrome; acute lung injury; keratinocyte growth factor; lipopolysaccharide; clinical trial
17.  Detection of Streptococcus pneumoniae Antigen in Bronchoalveolar Lavage Fluid Samples by a Rapid Immunochromatographic Membrane Assay 
Journal of Clinical Microbiology  2005;43(8):4037-4040.
We conducted a retrospective study to evaluate an immunochromatographic membrane test (ICT), applied to bronchoalveolar lavage (BAL) fluid samples obtained in patients with suspected pneumonia, for the detection of Streptococcus pneumoniae antigen. The NOW Streptococcus pneumoniae test was assessed on 96 BAL fluid samples. Sensitivity was tested in 20 samples obtained from patients diagnosed as having pneumococcal pneumonia (growth of S. pneumoniae in blood cultures and/or in BAL fluid samples of ≥104 CFU/ml). Specificity was tested in BAL fluid samples of nonpneumococcal etiology (n = 41) and in samples with no respiratory pathogen and a total bacterial count of <104 CFU/ml (n = 35). Using the ICT, pneumococcal antigen was detected in 29 (30.2%) BAL fluid samples, with a sensitivity of 95.0% (95% confidence interval [CI], 90.6% to 99.4%) and a specificity of 86.8% (95% CI, 80.1% to 93.8%). The ICT was easy to perform and revealed unequivocal and reproducible results. No interference was observed with high cell counts, red blood cells, or elevated protein levels. Four out of 10 false-positive readings occurred in samples with S. pneumoniae counts below the 104 CFU/ml threshold limit of pneumonia. In BAL fluid samples obtained after pneumococcal bacteremia, positive test results were found for up to 35 days after bacteremia. The ICT test applied to BAL fluid specimens is reproducible and accurate in the diagnosis of pneumococcal antigen. Further studies are required to establish the impact of the ICT on patient care.
PMCID: PMC1233991  PMID: 16081947
18.  Airways inflammation in subjects with chronic bronchitis who have never smoked. 
Thorax  1994;49(12):1211-1216.
BACKGROUND--Smoking is the single most common cause of chronic bronchitis but the disease can also occur in non-smokers. Alterations in the lung responsible for the disease, such as oxidant/antioxidant and protease/antiprotease imbalance, have been investigated in smokers. The aim of our study was to evaluate local cellular and soluble factors (albumin, immunoglobulins, proteases, alpha 1-antitrypsin, and transferrin) that may be involved in the development of chronic bronchitis in subjects who have never smoked. METHODS--Sixteen clinically stable patients with chronic bronchitis who had never been smokers were studied and 17 healthy non-smokers served as controls. All subjects underwent bronchoalveolar lavage (BAL). Total and differential cell counts and concentrations of the main proteins (albumin, immunoglobulins, complement fractions, alpha 1-antitrypsin, and transferrin) were measured. Elastase-like activity was assessed in cells and supernatants. To estimate the oxidant burden the release of superoxide anion (O2-) from native cell populations was evaluated. RESULTS--Recovery of BAL fluid was reduced in older individuals in both the chronic bronchitis and control groups. There was no difference in total cell count, but neutrophil percentage count was higher in those with chronic bronchitis (median (range) 3.5 (1.6-14.2)) than in controls (1.3 (0.5-3.7)). These differences were most pronounced in the first recovery, representative of the bronchial lavage. There was no difference in bronchial epithelial cells. Total proteins and albumin levels were comparable and IgG, IgA, IgM, C3, C4, transferrin and alpha 1-antitrypsin values standardised to albumin did not show any significant differences. No differences in elastase-like levels in supernatants were detected. In cell lysates elastase-like activity x 10(7) cells (macrophages+neutrophils) was increased in patients with chronic bronchitis (0.25 (0.06-4.3) compared with controls 0.08 (0.03-0.9) micrograms PPEeq). The release of O2- both at baseline and after opsonised zymosan phagocytosis did not show any differences. Correlation analysis between FEV1 and BAL fluid data showed a negative correlation only with neutrophils/ml. CONCLUSIONS--Clinically stable non-smokers with chronic bronchitis show no alterations of local immune components, oxidant burden, and free elastase-like activity in BAL fluids, while the content of elastase-like activity in phagocytic cells is increased. As in smokers, bronchial neutrophilia is the most significant cellular modification which correlates with the degree of airflow obstruction.
PMCID: PMC475325  PMID: 7878554
19.  Release of B cell-activating factor of the TNF family in bronchoalveolar lavage from Behçet disease with pulmonary involvement 
Pulmonary artery aneurysms, arterial and venous thrombosis, pulmonary infarction, recurrent pneumonia, bronchiolitis obliterans organized pneumonia, and pleurisy are the main features of pulmonary involvement in Behçet disease. The objective of this study was to investigate the production of B-cell-activating factor of the TNF family (BAFF), an important regulator of B-cell survival and immunoglobulin class-switch recombination, in bronchoalveolar lavage (BAL) fluid from BD patients having pulmonary manifestation. Bronchoalveolar lavage (BAL) was performed in 15 BD patients with pulmonary manifestation and 18 BAL from healthy controls. Concentrations of B-cell-active cytokines, including BAFF, IL-6 and IL-13, were measured by using specific ELISA and cytometric bead array assays. Levels of BAFF protein were significantly increased in BAL fluid from active BD (109 ± 21.78 pg/mL) compared with those oh healthy controls (4.83 ± 1.75 pg/mL; p < 0.0001). In the BAL fluid, BAFF levels were significantly correlated with absolute numbers of total cells (r = 0.823; p < 0.0001), lymphocytes (r = 0.709; p < 0.0001), neutrophils (r = 0.809; p < 0.0001) and macrophages (r = 0.742; p < 0.0001). Normalization to albumin indicated that BAFF production occurred locally in the airways. BAFF levels were also significantly correlated with the other B-cell-activating cytokines IL-6 (r = 0.882, p < 0.001) and IL-13 (r = 0.659, p < 0.001). The antigen-induced production of BAFF in the lung of active BD with pulmonary manifestations might contribute to immunoglobulin synthesis by B-cells. The cells residing in the lung might affect each other through BAFF.
PMCID: PMC2952096  PMID: 20716936
B cell-activating factor of the TNF family; Behçet disease; IL-6; IL-13; bronchoalveolar lavage
20.  Efficacy of BAL5788, a Prodrug of Cephalosporin BAL9141, in a Mouse Model of Acute Pneumococcal Pneumonia 
BAL5788 is a water-soluble prodrug of BAL9141, a new broad-spectrum cephalosporin with high levels of in vitro activity against methicillin- and vancomycin-resistant staphylococci and penicillin-resistant streptococci. In plasma BAL5788 is rapidly converted to BAL9141. We studied the activity of BAL5788 in a mouse model of acute pneumococcal pneumonia. Leukopenic female Swiss albino mice were challenged intratracheally with 107 CFU of clinical Streptococcus pneumoniae strains P-52181 (Pens Cros Ctxs), P-15986 (Penr Cros Ctxs), P-40422 (Penr Cror Ctxr), and P-40984 (Penr Cror Ctxr). Infected mice received subcutaneous (s.c.) injections of BAL5788 or ceftriaxone starting 3 h after pneumococcal challenge. Uninfected nonleukopenic mice received single s.c. doses of BAL5788 to determine the BAL9141 concentration-time profiles in serum and lungs. Untreated control mice died within 5 days postinfection. Ten-day cumulative survival rates for infected mice receiving BAL5788 (total daily doses of BAL9141 equivalents, 2.1 to 75 mg/kg of body weight) ranged from 57 to 100%, whereas with ceftriaxone (total daily doses, 10 to 400 mg/kg), the survival rates varied between 13 and 100%. In mice infected with P-15986, the survival rates achieved with BAL5788 (BAL9141 equivalent, 8.4 mg/kg) and those achieved with ceftriaxone (50 mg/kg) were significantly different (93 versus 13%; P < 0.0001) in favor of BAL5788; the outcomes of the trials with all other strains were not significantly different between the two antibiotics, but markedly lower doses of BAL5788 than ceftriaxone were required to obtain similar survival rates. Pharmacokinetic data showed that BAL9141 was effective against the four pneumococcal strains tested at very low values of the time above the MIC (T > MIC), which ranged from 9 to 18% of the dosing interval, whereas the values of T > MICs for ceftriaxone ranged from 30 to 50% of the dosing interval.
PMCID: PMC375270  PMID: 15047508
21.  Intrapulmonary protein leakage in immunocompromised children and adults with pneumonia 
Thorax  1999;54(5):432-436.
BACKGROUND—Pulmonary infections are associated with an increase in capillary permeability but information regarding age related differences in the local inflammatory response is lacking. To quantify the degree of capillary leakage during inflammation, the concentrations of the plasma proteins albumin, α1-antitrypsin, α2-macroglobulin and the locally produced proteins elastase, myeloperoxidase, lactoferrin and fibronectin were studied in the bronchoalveolar lavage (BAL) fluid of immunosuppressed children and adults with pneumonia.
METHODS—Sixteen children aged 2-16 years and 15 adults who developed pneumonia while receiving immunosuppressive therapy for haematological malignancies were included in the study. Bronchoalveolar lavage was performed via a flexible bronchoscope with three aliquots of 1 ml/kg body weight in children and 200 ml in adults. Protein concentrations in BAL fluid were determined using highly sensitive immunoluminometric assays.
RESULTS—Despite considerable variability, the median concentrations of all proteins in BAL fluid were significantly higher in both patient populations than in previously collected age adjusted reference values. The concentrations of serum derived proteins were significantly higher in children with pneumonia than in adult patients. In contrast, no differences were observed between the two groups for locally produced proteins.
CONCLUSIONS—These data suggest that the degree of protein exudation is more pronounced in immunosuppressed children with pneumonia than in adults in a similar clinical situation. This is in agreement with our studies in healthy individuals and may reflect a greater permeability of the alveolar-capillary membrane in children, regardless of disease status.

PMCID: PMC1763773  PMID: 10212109
22.  Evaluation of albumin as a reference marker of dilution in bronchoalveolar lavage fluid from asthmatic and control subjects. 
Thorax  1993;48(5):518-522.
BACKGROUND--Standardised expression of results of bronchoalveolar lavage (BAL) is problematical in the absence of a validated "denominator" of epithelial lining fluid dilution. The suitability of albumin in BAL fluid has been investigated in groups of clinically stable asthmatic and control subjects. METHODS--Absolute levels of albumin in BAL fluid were measured in a preliminary study of 21 asthmatic and 10 control subjects. In a more complex study designed to investigate the origin of albumin sampled at BAL in nine asthmatic and seven control subjects, radiolabelled albumin was injected intravenously five minutes before BAL. RESULTS--In the preliminary study levels of albumin in BAL fluid were very similar, with a geometric mean value of 44 (95% CI 35-54) micrograms/ml BAL supernatant for the asthmatic subjects and 41 (95% CI 33-52) micrograms/ml for the controls. The majority of control and asthmatic subjects in the radiolabel study exhibited minimal flux of albumin from the circulation into the BAL aspirate. This finding was not uniform, however, and in a third of the asthmatic subjects an albumin flux equivalent to > 20% of the measurable albumin was found in two or more aliquots of a 3 x 60 ml lavage. CONCLUSIONS--The results of this investigation into the source of albumin sampled at BAL suggest that, in general, albumin would be a reasonable reference solute for normalising the degree of dilution of BAL fluid in the groups studied. The origin of albumin was not always restricted to the bronchopulmonary segment under investigation, however, with significant leakage from the blood compartment in some individuals despite the consistency of absolute levels observed in the preliminary study.
PMCID: PMC464506  PMID: 8322239
23.  Regulation of vascular endothelial growth factor bioactivity in patients with acute lung injury 
Thorax  2005;60(2):153-158.
Methods: Forty one patients with ARDS, 12 at risk of developing ARDS, and 16 normal controls were studied. Bioactive VEGF, total VEGF, and sVEGFR-1 were measured by ELISA in plasma and bronchoalveolar lavage (BAL) fluid. Reverse transcriptase polymerase chain reaction for sVEGFR-1 was performed on BAL cells.
Results: sVEGFR-1 was detectable in the BAL fluid of 48% (20/41) of patients with early ARDS (1.4–54.8 ng/ml epithelial lining fluid (ELF)) compared with 8% (1/12) at risk patients (p = 0.017) and none of the normal controls (p = 0.002). By day 4 sVEGFR-1 was detectable in only 2/18 ARDS patients (p = 0.008). Patients with detectable sVEGFR-1 had lower ELF median (IQR) levels of bioactive VEGF than those without detectable sVEGFR-1 (1415.2 (474.9–3192) pg/ml v 4761 (1349–7596.6) pg/ml, median difference 3346 pg/ml (95% CI 305.1 to 14711.9), p = 0.016), but there was no difference in total VEGF levels. BAL cells expressed mRNA for sVEGFR-1 and produced sVEGFR-1 protein which increased following incubation with tumour necrosis factor α.
Conclusion: This study shows for the first time the presence of sVEGFR-1 in the BAL fluid of patients with ARDS. This may explain the presence of reduced bioactive VEGF in patients early in the course of ARDS.
PMCID: PMC1747283  PMID: 15681505
24.  Cell-associated bacteria in the human lung microbiome 
Microbiome  2014;2:28.
Recent studies have revealed that bronchoalveolar lavage (BAL) fluid contains previously unappreciated communities of bacteria. In vitro and in vivo studies have shown that host inflammatory signals prompt bacteria to disperse from cell-associated biofilms and adopt a virulent free-living phenotype. The proportion of the lung microbiota that is cell-associated is unknown.
Forty-six BAL specimens were obtained from lung transplant recipients and divided into two aliquots: ‘whole BAL’ and ‘acellular BAL,’ the latter processed with a low-speed, short-duration centrifugation step. Both aliquots were analyzed via bacterial 16S rRNA gene pyrosequencing. The BAL specimens represented a wide spectrum of lung health, ranging from healthy and asymptomatic to acutely infected. Bacterial signal was detected in 52% of acellular BAL aliquots, fewer than were detected in whole BAL (96%, p ≤ 0.0001). Detection of bacteria in acellular BAL was associated with indices of acute infection [BAL neutrophilia, high total bacterial (16S) DNA, low community diversity, p < 0.01 for all] and, independently, with low relative abundance of specific taxonomic groups (p < 0.05). When whole and acellular aliquots from the same bronchoscopy were directly compared, acellular BAL contained fewer bacterial species (p < 0.05); whole and acellular BAL similarity was positively associated with evidence of infection and negatively associated with relative abundance of several prominent taxa (p < 0.001). Acellular BAL contained decreased relative abundance of Prevotella spp. (p < 0.05) and Pseudomonas fluorescens (p < 0.05).
We present a novel methodological and analytical approach to the localization of lung microbiota and show that prominent members of the lung microbiome are cell-associated, potentially via biofilms, cell adhesion, or intracellularity.
PMCID: PMC4158729  PMID: 25206976
Lung microbiome; Bronchoalveolar lavage; 16S; Pyrosequencing; Pneumonia
25.  Fibroblast chemotactic response elicited by native bronchoalveolar lavage fluid from patients with fibrosing alveolitis. 
Thorax  1993;48(7):736-742.
BACKGROUND--In fibrosing alveolitis activation of lung fibroblasts is the decisive event in the pathogenetic sequence leading to pulmonary fibrosis. Fibroblast stimulating activity was measured in bronchoalveolar lavage (BAL) fluid to assess its relationship to the activity of fibrosing alveolitis. METHODS--Nine control subjects and 40 patients with fibrosing alveolitis caused by idiopathic pulmonary fibrosis (n = 22) or pulmonary involvement in systemic sclerosis (n = 18) were studied. All patients were followed up by lung function testing for a minimum of six months (mean (SE) 13.3 (1.4) months). Twenty five patients received immunosuppressive therapy and 15 refused. At the beginning of follow up BAL was performed and, as a possible indicator of fibroblast stimulating mediators within the lungs, chemotactic migration of cultured human fibroblasts elicited by native BAL fluid was measured in Boyden-type chambers and expressed as a percentage of the chemoattractant effect of 25 ng/ml platelet derived growth factor. The procollagen III peptide level in BAL fluid served as a marker for collagen synthesis. RESULTS--Chemoattractant activity was elevated in the patients with idiopathic pulmonary fibrosis and systemic sclerosis compared with the control group, (mean (SE) 56.4% (8.5%)) and 72.3% (16.3%) v 12.6% (4.0%). Chemoattractant activity was inversely correlated with total lung capacity (TLC) (r = -0.45) and with vital capacity (VC) (r = -0.33). Procollagen III peptide concentrations in BAL fluid and chemoattractant activity were not significantly correlated. For further evaluation chemoattractant activity of 36% (mean value of controls +2 SD) was used to separate normal (< 36%) from elevated (> or = 36%) activity. At the end of follow up, untreated patients with high chemoattractant activity (> or = 36%) showed a significant reduction of VC, TLC, and exercise arterial oxygen tension (PaO2) and a small decrease in carbon monoxide transfer factor (TLCO), whereas a significant improvement in VC, TLC, and TLCO and a small increase of exercise PaO2 occurred in treated patients with high chemoattractant activity. Patients with low chemoattractant activity (< 36%) showed no consistent change in lung function measurements, irrespective of treatment. In contrast, lung function results and differential cell counts in BAL fluid failed to identify progressive disease. CONCLUSIONS--In patients with fibrosing alveolitis the chemoattractant activity of BAL fluid seems to be an independent indicator of lung fibroblast stimulating activity providing relevant information about disease activity, and may help to improve the clinical management of these patients.
PMCID: PMC464660  PMID: 8153923

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