A new strategy to create ordered deletions for rapid nucleotide sequencing

A method is described for generating ordered deletions using previously published techniques but a new strategy. This method is simpler than the published ones and has many advantages. Target DNA is cloned in both orientations into one of the unique restriction enzyme sites adjacent to the complementary region of the commercially available primers in bacteriophage M13. Ordered unidirectional deletions are created using BAL 31 nuclease and religating into M13 vector DNA without the need of purifying BAL 31-digested DNA from a gel.     

Site-specific DNA splicing: a general procedure for the creation of a restriction site at a predetermined position in a DNA sequence

A method is described for creating any of a wide array of restriction sites at a predetermined position in a known DNA sequence. The method utilizes the exonuclease activity of BAL 31 and a specially designed bifunctional oligodeoxynucleotide linker. The desired restriction site is generated when the linker is ligated to those BAL 31-digested DNA fragments which end with the target sequence. The proper ligation product is then identified by a highly specific hybridization procedure. The method is versatile and specific and is especially useful in the isolation of functional elements of a gene.     

Measurement of cefuroxime in human bronchoalveolar lavage fluid by high-performance liquid chromatography after solid-phase extraction

A sensitive and selective method for the determination of cefuroxime in bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography (HPLC) with UV detection at 280 nm after solid-phase extraction with C₁₈ cartridges was developed. A Waters symmetry C₁₈ column was used and the mobile phase was acetonitrile-0.05 M ammonium phosphate buffer (pH 3.2) (15:85, v/v). The method enabled the determination of cefuroxime at concentrations below 100 ng/ml, with a linear calibration curve at concentrations of 5–100 ng/ml for 400 μl of BAL. The intra- and inter-assay coefficient of variations for 10, 40 and 80 ng/ml were between 5.3 and 8.9%. Analytical recoveries were between 92.7 and 106.2%. The detection limit was 1 ng/ml at a signal-to-noise ratio of 3:1 using 400 μl of BAL. The method was successfully used for the analysis of BAL fluid from patients after oral administration of 500 mg cefuroxime axetil twice daily.     

A comparison of modified methenamine silver and toluidine blue stains for the detection of Pneumocystis carinii in bronchoalveolar lavage specimens from immunosuppressed patients.

Pneumonia due to Pneumocystis carinii is an increasingly frequent occurrence; a prompt, accurate diagnosis is important to successfully manage this infection. Methenamine silver and toluidine blue stainings of material recovered by bronchoalveolar lavage (BAL) have been the most widely employed techniques for detecting Pneumocystis organisms. The value of these two staining techniques for the detection of Pneumocystis organisms was prospectively compared in 220 BAL specimens obtained from 186 immunosuppressed individuals. The patients included those with disease-related and therapy-related immunosuppression; half of the BAL specimens came from organ transplant recipients. The results indicate that neither method is superior to the other and that both will correctly identify almost all Pneumocystis infections if a sufficient aliquot of BAL material is examined systematically by trained individuals. The toluidine blue method is somewhat simpler to perform, however. The reasons for discrepant results between the two staining methods were analyzed.     

High-performance liquid chromatographic method for the determination of budesonide in bronchoalveolar lavage of asthmatic patients

A simple, sensitive and selective method for the determination of budesonide in bronchoalveolar lavage (BAL) using high-performance liquid chromatography (HPLC) with UV detection was developed. BAL samples were extracted twice with methylene chloride, the extracts were centrifuged and the organic laeer separated and dried under nitrogen. The samples were reconstituted in the mobile phase and 80 μl were injected on to a Spherisorb ODS column with UV absorbance detection at 250 nm. The mobile phase was methanol-aqueius buffer (69:31, v/v). Inter-assay coefficients of variation were measured at 7.81 and 500 ng/ml with ranges of 0.89–7.31%. Average recoveries were 97% or greater. This method was successfully implemented for the analysis of BAL from asthmatics, in order to establish the amount of budesonide available to the lung and to investigate the efficacy of inhaler systems. Patients (n = 9) inhaled four puffs of 200 μg of budesonide and BAL was perforned 10 min after the last inhalation. Only four BAL out of the nine presented detectable amounts of budesonide. The concentrations in BAL in these four patients were 13.44–84.18 ng/ml, corresponding to total anounts of 0.847–7.997 μg.     

用聚合酶链反应（PCR）检测支气管肺泡灌洗（BAL）液中的嗜肺军团杆菌

本文研究了由嗜肺军团杆菌的巨噬细胞感染性增效子（ｍｉｐ）基因设计的一对引物,用ＰＣＲ扩增嗜肺军团杆菌３、５、７、８血清型的４个标准菌株的特异ＤＮＡ序列,研究了用该引物扩增ＢＡＬ液中嗜肺军团菌特异ＤＮＡ序列的方法、灵敏度及特异性。结果表明：用上述引物扩增嗜肺军团菌４个标准菌株的ＤＮＡ,均可得到２０７ｂｐ的特异扩增产物,ＢＡＬ液中的军团菌经离心及裂解液裂解后,可直接进行ＤＮＡ扩增,当ＢＡＬ与液中军团菌量为２×１０３ＣＦＵ／ｍｌ时,即可检测出特异扩增带（电泳法）,除军团菌外,其它受试细菌均无此特异性扩增,用本法对４２例临床非典型肺炎患者的ＢＡＬ液进行嗜肺军团菌的检测,在４２份嗜肺军团菌培养均为阴性的ＢＡＬ液中,其中一例ＰＣＲ检测军团菌为阳性。本研究提示：用ＰＣＲ检测ＢＡＬ液中的军团菌是可行的,并有快速、灵敏、特异之忧点。     

Two‐sample nonparametric likelihood inference based on incomplete data with an application to a pneumonia study

The clinical pulmonary infection score (CPIS) and bronchoalveolar lavage (BAL) are important diagnostic variables of pneumonia for forcefully ventilated patients who are susceptible to nosocomial infection. Because of its invasive nature, BAL is performed for patients only if the CPIS is greater than a certain threshold value. Thus, CPIS and BAL are closely related, yet BAL values are substantially missing. In a randomized clinical trial, the control and oral treatment groups were compared based on the outcomes from these procedures. Because of the relevance of both outcomes with respect to evaluating the efficacy of treatments, we propose and examine a nonparametric test based on these outcomes, which employs the empirical likelihood methodology. While efficient parametric methods are available when data are observed incompletely, performing appropriate goodness‐of‐fit tests to justify the parametric assumptions is difficult. Our motivation is to provide an approach based on no particular distributional assumption, which enables us to use all observed bivariate data, whether completed or not in an approximate likelihood manner. A broad Monte Carlo study evaluates the asymptotic properties and efficiency of the proposed method based on various sample sizes and underlying distributions. The proposed technique is applied to a data set from a pneumonia study demonstrating its practical worth.     

Structure of human milk bile salt activated lipase

The structure and some functional sites of human milk bile salt activated lipase (BAL) were studied by cDNA cloning and chemical analysis of the enzyme. Eighteen cDNA clones of human BAL were identified from lactating human breast cDNA libraries in lambda gt11 and lambda gt10 with antibody and synthetic oligonucleotides as probes. The sequence of four clones was sufficient to construct a 3018-bp BAL cDNA structure. This sequence codes for an open reading frame of 742 amino acid residues. There is a putative signal sequence of 20 residues which is followed by the amino-terminal sequence of BAL, and the mature BAL contains 722 amino acid residues. The cDNA sequence also contains a 678-base 5'-untranslated sequence, a 97-base 3'-untranslated region, and a 14-base poly(A) tail. The sequence of a 1.8-kbp insert of clone G10-4A differs from that of the other cDNA in that it contains a deletion of 198 bases (1966-2163) corresponding to 66 amino acid residues. By use of BAL cDNA as probe, it was found that the major molecular species of BAL mRNA in human mammary gland HBL-100 cells had a size of 2.9 kb and two minor species had sizes of 3.8 and 5.1 kb by Northern blot analyses. The deduced BAL protein structure contains in the carboxyl-terminal region 16 repeating units of 11 amino acids each. The repeating units have the basic structure Pro-Val-Pro-Pro-Thr-Gly-Asp-Ser-Gly-Ala-Pro with only minor substitutions. The amino acid sequence of human BAL is related to that of pancreatic lysophospholipase, cholesterol esterase, cholinesterase, acetylcholinesterase, and thyroglobulin. Ten of the 14 cyanogen bromide fragments of diisopropyl fluorophosphate inhibited human milk BAL were isolated, determined for N-terminal sequences, analyzed for amino sugars, and tested for some functional properties. These chemical studies established that the active site of human milk BAL is located at serine-194, the N-glycosylation site is present at asparagine-187, the O-glycosylation region is in the 16 repeating units near the C-terminus, and the heparin binding domain is in the N-terminal region. We have also determined the location of disulfide bridges as Cys64-Cys80 and Cys246-Cys257. The cyanogen bromide cleavage and the partial sequencing of CNBr peptides also confirmed the location of methionines in the polypeptide chain as well as the deduced cDNA sequence of BAL.     

Increased sensitivity of a direct fluorescent antibody test for Legionella pneumophila in bronchoalveolar lavage samples by immunomagnetic separation based on BioMags

In the present study, immunomagnetic separation of Legionella pneumophila from mock bronchoalveolar lavage (BAL) fluid samples, which were artificially spiked with L. pneumophila, and culture positive patient BAL fluid samples, was achieved using BioMags (superparamagnetic particles) loaded with purified rabbit immunoglobulin G specific for L. pneumophila. Bacteria binding onto BioMag-immunomatrix were directly stained with a L. pneumophila species-specific DFA reagent and examined under a fluorescence microscope. BioMag-based immunomagnetic separation (BIMS) followed by DFA staining (BIMS-DFA) could correctly identify all the 20 (100%) BAL samples which were spiked with low numbers (2x10(2) CFU) of L. pneumophila. Cultures could be recovered from 15 (75%) of these 20 spiked BAL samples, 5 (25%) of the samples failed to yield positive cultures. Both culture and BIMS-DFA methods showed 100% positive results when spiked BAL samples containing high bacterial load (10(4) CFU) were tested. The findings with true patient culture positive BAL specimens which were examined retrospectively indicated that BIMS-DFA is significantly more sensitive for detecting L. pneumophila than conventional cytospin method of DFA staining (cytospin-DFA). Out of the 25 culture positive BAL specimens tested, 7 (28%) proved negative by cytospin-DFA whereas BIMS-DFA correctly detected all the 25 (100%) specimens. It is suggested that the BIMS-DFA procedure increases the sensitivity of DFA testing for L. pneumophila in large volume samples such as BAL fluids.     

Determination of free ertapenem in plasma and bronchoalveolar lavage by high-performance liquid chromatography with ultraviolet detection

A sensitive assay for the determination of unbound ertapenem in human plasma and bronchoalveolar lavage (BAL) was developed using ultrafiltration of plasma and BAL samples. A rapid HPLC method was used with ultraviolet detection set at a wavelength of 305 nm and a separation on a Prontosil AQ C18 column, with imipenem used as internal standard. This assay was linear over the concentration range of 0.5-100 microg/mL and 0.25-50 microg/mL in plasma and BAL, respectively. Limits of detection and quantitation were respectively 0.05 and 0.25 microg/mL. Validation data for accuracy and precision were CV<2.48 and 8.25%, accuracy in the range 98.1-104.2% and 102.2-108.4%, respectively, for intra and inter-day.     

Differential determination of phospholipase A(2) and PAF-acetylhydrolase in biological fluids using fluorescent substrates

The purpose of the present study was the development and evaluation of a fluorimetric method for the screening and differential determination of phospholipase A(2) and PAF-acetylhydrolase in bronchoalveolar lavage (BAL) fluid and serum. Phospholipase A(2) was determined using C(12)-NBD-PC in the presence of Ca(2+), from the slope of the fluorescence enhancement due to the formation of C(12)-NBD-fatty acid. PAF-acetylhydrolase was determined using C(6)-NBD-PC, from the slope of the curve due to C(6)-NBD-fatty acid formation in the absence of Ca(2+). The results were confirmed after TLC analysis. The method's selectivity was evaluated by comparing to radiometric measurements. Light scattering did not interfere and inner filter effects was not observed under our experimental conditions. The effects of pH, temperature, and Ca(2+) were investigated. Protein caused an increase in the background fluorescence of both NBD-PCs. The standard curves of both NBD-fatty acids exhibited the same slope. Linearity extended at least up to 4. 5 nmoles per ml of reaction mixture at the normal pH 7.4. The fluorescence of the NBD-fatty acids remained stable for increasing concentrations of BAL fluid and serum and for BSA up to 100 microg/ml of reaction mixture. Porcine pancreatic PLase A(2) showed preference for C(12)-NBD-PC in the presence of Ca(2+), while without Ca(2+), serum PAF-AcH hydrolyzed only C(6)-NBD-PC. The method is highly sensitive, accurate, and reproducible and can be applied for the differential determination of phospholipase A(2) and PAF-acetylhydrolase activities in BAL fluid and serum.     

Adoptive transfer of acute lung injury

In this study, we describe a novel adoptive transfer protocol to study acute lung injury in the rat. We show that bronchoalveolar lavage (BAL) cells isolated from rats 5 h after intratracheal administration of lipopolysaccharide (LPS) induce a lung injury when transferred to normal control recipient rats. This lung injury is characterized by increased alveolar-arterial oxygen difference and extravasation of Evans blue dye (EBD) into lungs of recipient rats. Recipient rats receiving similar numbers of donor cells isolated from healthy rats do not show adverse changes in the alveolar-arterial oxygen difference or in extravasation of EBD. The adoptive transfer-induced lung injury is associated with increased numbers of neutrophils in the BAL, the levels of which are similar to the numbers observed in BAL cells isolated from rats treated for 5 h with LPS. As an indicator of BAL cell activation, donor BAL cell inducible nitric oxide synthase (iNOS) expression was compared with BAL cell iNOS expression 48 h after adoptive transfer. BAL cells isolated 5 h after LPS administration expressed iNOS immediately after isolation. In contrast, BAL cells isolated 48 h after adoptive transfer did not express iNOS immediately after isolation but expressed iNOS following a 24-h ex vivo culture. These findings indicate that the activation state of donor BAL cells differs from BAL cells isolated 48 h after adoptive transfer, suggesting that donor BAL cells may stimulate migration of new inflammatory cells into the recipient rats lungs.     

Evaluation methods and design for bioartificial liver based on perfusion model

A bioartificial liver (BAL) is a medical device entrapping living hepatocytes or immortalized cells derived from hepatocytes. Many efforts have already been made to maintain the functions of the hepatocytes in a BAL device over a long term. However, there is still some uncertainty as to their efficacy, and their limitations are unclear. Therefore, it is important to quantitatively evaluate the metabolic functions of a BAL. In previous studies onin vitro BAL devices, two test methods, an initial bolus loading and constant-rate infusion plus initial bolus loading, were theoretically carried out to obtain physiologic data on drugs. However, in the current study, the same two methods were used as a perfusion model and derived the same clearance characterized by an interrelationship between the perfusate flow rate and intrinsic clearance. The interrelationship indicated that the CL increased with an increasing perfusate flow rate and approached its maximum value,i.e. intrinsic clearance. In addition, to set up anin vivo BAL system, the toxic plateau levels in the BAL system were calculated for both series and parallel circuit models. The series model had a lower plateau level than the parellel model. The difference in the toxic plateau levels between the parallel and series models increased with an increasing number of BAL cartridges.     

Human bronchoalveolar lavage: biofluid analysis with special emphasis on sample preparation

Respiratory diseases are an important health problem throughout the world. Whether caused by industrial pollutants, infections, smoking, cancer or metabolic diseases, damage to the lungs and airways often lead to morbidity or death. Bronchoalveolar lavage (BAL) obtained by fiber-optic bronchoscopy is a biofluid mirroring the expression of normally secreted pulmonary proteins and the products of activated cells and destructive processes. The characterization of the proteome within this compartment provides an opportunity to establish temporal and prognostic indicators of airway disease. The objective of this study was to develop methods of analysis of BAL samples, which achieved the highest level of annotation of the expression map of this proteome. We have optimized the process of sample preparation after investigating a variety of techniques including dialysis, ultramembrane filtration, precipitation and gel filtration. We have further studied methods to remove albumin from BAL in order to unmask proteins hidden on two-dimensional gels. In a pilot application of the method, BAL protein profiles obtained from healthy nonsmokers and smokers at risk for developing chronic obstructive pulmonary disease showed distinct differences.     

Cytochrome b5 and cytokeratin 17 are biomarkers in bronchoalveolar fluid signifying onset of acute lung injury

Acute lung injury (ALI) is characterized by pulmonary edema and acute inflammation leading to pulmonary dysfunction and potentially death. Early medical intervention may ameliorate the severity of ALI, but unfortunately, there are no reliable biomarkers for early diagnosis. We screened for biomarkers in a mouse model of ALI. In this model, inhalation of S. aureus enterotoxin A causes increased capillary permeability, cell damage, and increase protein and cytokine concentration in the lungs. We set out to find predictive biomarkers of ALI in bronchoalveolar lavage (BAL) fluid before the onset of clinical manifestations. A cutting edge proteomic approach was used to compare BAL fluid harvested 16 h post S. aureus enterotoxin A inhalation versus BAL fluid from vehicle alone treated mice. The proteomic PF 2D platform permitted comparative analysis of proteomic maps and mass spectrometry identified cytochrome b5 and cytokeratin 17 in BAL fluid of mice challenged with S. aureus enterotoxin A. Validation of cytochrome b5 showed tropic expression in epithelial cells of the bronchioles. Importantly, S. aureus enterotoxin A inhalation significantly decreased cytochrome b5 during the onset of lung injury. Validation of cytokeratin 17 showed ubiquitous expression in lung tissue and increased presence in BAL fluid after S. aureus enterotoxin A inhalation. Therefore, these new biomarkers may be predictive of ALI onset in patients and could provide insight regarding the basis of lung injury and inflammation.     

Detection of Aspergillus fumigatus hyphae in respiratory secretions by membrane filtration, fluorescent labelling and laser scanning

The detection of Aspergillus fumigatus hyphae in bronchoalveolar lavage fluid (BAL) and sputum is diagnostically useful in patients at risk of invasive aspergillosis. We report a dedicated enzymatic-chemical sample pretreatment that allows the application of a previously described solid phase cytometry (SPC) method for detection of A. fumigatus hyphae in sputum and BAL samples. Non-specific detection of fungal hyphae by SPC is based on a 'viability' staining using carboxyfluorescein diacetate. For a specific detection of A. fumigatus hyphae by SPC, viability staining is combined with a pre-incubation at 45 degrees C, immunofluorescent labelling and microscopic recognition of the characteristic hyphal morphology. Low numbers of A. fumigatus hyphae (2-10 hyphae/sample) have now been demonstrated in spiked sputum using the non-specific and specific staining in 2.5 and 8.5 h, respectively.     

Analysis of Trace Elements in Bronchoalveolar Lavage of Patients with Diffuse Lung Diseases

Airborne trace elements are implicated in the etio-pathogenesis of a large number of pulmonary diseases. The aim of this study was to evaluate the reliability and effectiveness of direct determination of Cd, Cr, Cu, Fe, Mn, Ni, Pb, V, and Zn concentrations in bronchoalveolar lavage (BAL) samples from patients with sarcoidosis, idiopathic pulmonary fibrosis, and Langerhans cell histiocytosis and healthy (smoking and non-smoking) controls. A total of 44 individuals were recruited among sarcoidosis, idiopathic pulmonary fibrosis, and Langerhans cell histiocytosis patients and healthy (smoking and non-smoking) controls. Average Mn concentrations in BAL from patients were 45% lower than in controls (p < 0.01) and remarkable decreases in average concentrations of Cr, Ni and Zn were also found in BAL from patients with idiopathic pulmonary fibrosis and Langerhans cell histiocytosis. As these diseases are characterized by the enhanced activation of certain immunomodulatory cells and by generation of free radicals, the depressed Mn, Zn, Cr and Ni concentrations in BAL from patients may be due to oxidative stress. These preliminary results indicate that assessment of the elemental composition of BAL is a promising approach to study the pathogenesis of diffuse lung diseases and Langerhans cell histiocytosis.     

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.