Isolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy.
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ABSTRACT:
SUBMITTER: Wichmann C
PROVIDER: S-EPMC8405473 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:bronchoalveolar lavage fluid Metagenome
| PRJNA449183 | ENA
Project description:Extracellular vesicles (EVs) are membrane-bound particles that engage in inflammatory reactions by mediating cell-cell interactions. Previously, EVs have been isolated from the bronchoalveolar lavage fluid (BALF) of humans and rodents. The aim of this study was to investigate the number and size distribution of EVs in the BALF of asthmatic horses (EA, n = 35) and healthy horses (n = 19). Saline was injected during bronchoscopy to the right lung followed by manual aspiration. The retrieved BALF was centrifuged twice to remove cells and biological debris. The supernatant was concentrated and EVs were isolated using size-exclusion chromatography. Sample fractions were measured with nanoparticle tracking analysis (NTA) for particle number and size, and transmission electron microscopy and confocal laser scanning microscopy were used to visualize EVs. The described method was able to isolate and preserve EVs. The mean EV size was 247 ± 35 nm (SD) in the EA horses and 261 ± 47 nm in the controls by NTA. The mean concentration of EVs was 1.38 × 1012 ± 1.42 × 1012 particles/mL in the EA horses and 1.33 × 1012 ± 1.07 × 1012 particles/mL in the controls with no statistically significant differences between the groups. With Western blotting and microscopy, these particles were documented to associate with EV protein markers (CD63, TSG101, HSP70, EMMPRIN, and actin) and hyaluronan. Equine BALF is rich in EVs of various sizes, and the described protocol is usable for isolating EVs. In the future, the role of EVs can be studied in horses with airway inflammation.
| S-EPMC9255554 | biostudies-literature
Project description:BackgroundIt is now possible to comprehensively characterize the microbiota of the lungs using culture-independent, sequencing-based assays. Several sample types have been used to investigate the lung microbiota, each presenting specific challenges for preparation and analysis of microbial communities. Bronchoalveolar lavage fluid (BALF) enables the identification of microbiota specific to the lower lung but commonly has low bacterial density, increasing the risk of false-positive signal from contaminating DNA. The objectives of this study were to investigate the extent of contamination across a range of sample densities representative of BALF and identify features of contaminants that facilitate their removal from sequence data and aid in the interpretation of BALF sample 16S sequencing data.ResultsUsing three mock communities across a range of densities ranging from 8E+?02 to 8E+?09 16S copies/ml, we assessed taxonomic accuracy and precision by 16S rRNA gene sequencing and the proportion of reads arising from contaminants. Sequencing accuracy, precision, and the relative abundance of mock community members decreased with sample input density, with a significant drop-off below 8E+?05 16S copies/ml. Contaminant OTUs were commonly inversely correlated with sample input density or not reproduced between technical replicates. Removal of taxa with these features or physical concentration of samples prior to sequencing improved both sequencing accuracy and precision for samples between 8E+?04 and 8E+?06 16S copies/ml. For the lowest densities, below 8E+?03 16S copies/ml BALF, accuracy and precision could not be significantly improved using these approaches. Using clinical BALF samples across a large density range, we observed that OTUs with features of contaminants identified in mock communities were also evident in low-density BALF samples.ConclusionRelative abundance data and community composition generated by 16S sequencing of BALF samples across the range of density commonly observed in this sample type should be interpreted in the context of input sample density and may be improved by simple pre- and post-sequencing steps for densities above 8E+?04 16S copies/ml.
| S-EPMC6821041 | biostudies-literature
Project description:The long-term goal of our study is to identify chronic obstructive pulmonary disease (COPD)-related bronchoalveolar lavage fluid (BALF) nitroproteins to clarify COPD pathological mechanisms and to discover biomarkers of COPD. The goal of the present study was to detect the presence of, and potential roles of, nitroproteins in, human ex-smoker (without COPD) BALF samples. Nitroproteins were immunoprecipitated from two separate BALF samples, and digested with trypsin; and tryptic peptides were analyzed with matrix-assisted laser desorption/ionization (MALDI)-tandem mass spectrometry (MS/MS). Each MS/MS spectrum was composed of accumulated scans (n = 50-100). The MS/MS data were searched with BioWorks 2.0 TuboSequest in the SwissProt database to generate the amino acid sequence, which was evaluated manually. Eleven nitrotyrosine sites were identified in eight proteins, including progestin and adipoQ receptor family member III, zinc finger protein 432, proteasome subunit alpha type 2, NADH-ubiquinone oxidoreductase B14, slit homolog 1 protein, lysozyme, aldose 1-epimerase, and PTS system lactose-specific EIICB component. Each nitrotyrosine site was located within a specific protein domain and motif. Those identified nitrated proteins could be involved in multiple functional metabolic systems, including transcriptional regulation, mitochondrial complex, immune system, and energy metabolism.
| S-EPMC3295046 | biostudies-literature
Project description:Global-scale examination of protein phosphorylation in human biological fluids by phosphoproteomics approaches is an emerging area of research with potential for significant contributions towards discovery of novel biomarkers. In this pilot work, we analyzed the phosphoproteome in human bronchoalveolar lavage fluid (BAL) from nondiseased subjects. The main objectives were to assess the feasibility to probe phosphorylated proteins in human BAL and to obtain the initial catalog of BAL phosphoproteins, including protein identities and exact description of their phosphorylation sites. We used a gel-free bioanalytical workflow that included whole-proteome digestion of depleted BAL proteins, enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC), LC-MS/MS analyses with a linear ion trap mass spectrometer, and searches of a protein sequence database to generate a panel of BAL phosphoproteins and their sites of phosphorylation. Based on sequence-diagnostic MS/MS fragmentation patterns, we identified a collection of 36 phosphopeptides that contained 26 different phosphorylation sites. These phosphopeptides mapped to 21 phosphoproteins including, for example, vimentin, plastin-2, ferritin heavy chain, kininogen-1, and others. The characterized phosphoproteins have diverse characteristics in terms of cellular origin and biological function. To the best of our knowledge, results of this study represent the first description of the human BAL phosphoproteome.
| S-EPMC3446654 | biostudies-literature
Project description:Bronchoalveolar Lavage Fluid Pathogen Microbiology
| PRJEB72318 | ENA
Project description:BackgroundThe pathogenesis of invasive aspergillosis (IA) is still unknown, but its progression is rapid and mortality rate remains high. Bronchoalveolar lavage fluid (BALF) galactomannan (GM) analysis has been used to diagnose IA. This study is aimed at making an accurate estimate of the whole accuracy of BALF-GM in diagnosing IA.MethodsAfter a systematic review of the study, a bivariate meta-analysis was used to summarize the specificity (SPE), the sensitivity (SEN), the positive likelihood ratios (PLR), and the negative likelihood ratios (NLR) of BALF-GM in diagnosing IA. The overall test performance was summarized using a layered summary receiver operating characteristic (SROC) curve. Subgroup analysis was performed to explore the heterogeneity between studies.ResultsA total of 65 studies that are in line with the inclusion criteria were included. The summary estimates of BALF-GM analysis are divided into four categories. The first is the proven+probable vs. possible+no IA, with an SPE, 0.87 (95% CI, 0.85-0.98); SEN, 0.81 (95% CI, 0.76-0.84); PLR, 9.78 (5.78-16.56); and NLR, 0.20 (0.14-0.29). The AUC was 0.94. The BALF-GM test for proven+probable vs. no IA showed SPE, 0.88 (95% CI, 0.87-0.90); SEN, 0.82 (95% CI, 0.78-0.85); PLR, 6.56 (4.93-8.75); and NLR, 0.24 (0.17-0.33). The AUC was 0.93. The BALF-GM test for proven+probable+possible vs. no IA showed SPE, 0.82 (95% CI, 0.79-0.95); SEN, 0.59 (95% CI, 0.55-0.63); PLR, 3.60 (2.07-6.25); and NLR, 0.31 (0.15-0.61). The AUC was 0.86. The analyses for others showed SPE, 0.85 (95% CI, 0.83-0.87); SEN, 0.89 (95% CI, 0.86-0.91); PLR, 6.91 (4.67-10.22); and NLR, 0.18 (0.13-0.26). The AUC was 0.94.ConclusionsThe findings of this BALF-GM test resulted in some impact on the diagnosis of IA. The BALF-GM assay is considered a method for diagnosing IA with high SEN and SPE. However, the patients' underlying diseases may affect the accuracy of diagnosis. When the cutoff is greater than 1, the sensitivity will be higher.
| S-EPMC7723495 | biostudies-literature
Project description:BackgroundThe causative agents for the current national outbreak of electronic-cigarette, or vaping, product use-associated lung injury (EVALI) have not been established. Detection of toxicants in bronchoalveolar-lavage (BAL) fluid from patients with EVALI can provide direct information on exposure within the lung.MethodsBAL fluids were collected from 51 patients with EVALI in 16 states and from 99 healthy participants who were part of an ongoing study of smoking involving nonsmokers, exclusive users of e-cigarettes or vaping products, and exclusive cigarette smokers that was initiated in 2015. Using the BAL fluid, we performed isotope dilution mass spectrometry to measure several priority toxicants: vitamin E acetate, plant oils, medium-chain triglyceride oil, coconut oil, petroleum distillates, and diluent terpenes.ResultsState and local health departments assigned EVALI case status as confirmed for 25 patients and as probable for 26 patients. Vitamin E acetate was identified in BAL fluid obtained from 48 of 51 case patients (94%) in 16 states but not in such fluid obtained from the healthy comparator group. No other priority toxicants were found in BAL fluid from the case patients or the comparator group, except for coconut oil and limonene, which were found in 1 patient each. Among the case patients for whom laboratory or epidemiologic data were available, 47 of 50 (94%) had detectable tetrahydrocannabinol (THC) or its metabolites in BAL fluid or had reported vaping THC products in the 90 days before the onset of illness. Nicotine or its metabolites were detected in 30 of 47 of the case patients (64%).ConclusionsVitamin E acetate was associated with EVALI in a convenience sample of 51 patients in 16 states across the United States. (Funded by the National Cancer Institute and others.).
| S-EPMC7032996 | biostudies-literature
Project description:The analysis of airway fluid, as sampled by bronchoalveolar lavage (BAL), provides a minimally invasive route to interrogate lung biology in health and disease. Here, we used immunodepletion, coupled with gel- and label-free LC-MS/MS, for quantitation of the BAL fluid (BALF) proteome in samples recovered from human subjects following bronchoscopic instillation of saline, lipopolysaccharide (LPS) or house dust mite antigen into three distinct lung subsegments. Among more than 200 unique proteins quantified across nine samples, neutrophil granule-derived and acute phase proteins were most highly enriched in the LPS-exposed lobes. Of these, peptidoglycan response protein 1 was validated and confirmed as a novel marker of neutrophilic inflammation. Compared to a prior transcriptomic analysis of airway cells in this same cohort, the BALF proteome revealed a novel set of response factors. Independent of exposure, the enrichment of tracheal-expressed proteins in right lower lung lobes suggests a potential for constitutive intralobar variability in the BALF proteome; sampling of multiple lung subsegments also appears to aid in the identification of protein signatures that differentiate individuals at baseline. Collectively, this proof-of-concept study validates a robust workflow for BALF proteomics and demonstrates the complementary nature of proteomic and genomic techniques for investigating airway (patho)physiology.
| S-EPMC3822014 | biostudies-literature
Project description:BackgroundThe contribution of bronchoalveolar lavage fluid (BALF) microbiota and mycobiota to silicosis has recently been noticed. However, many confounding factors can influence the accuracy of BALF microbiota and mycobiota studies, resulting in inconsistencies in the published results. In this cross-sectional study, we systematically investigated the effects of "sampling in different rounds of BALF" on its microbiota and mycobiota. We further explored the relationship between silicosis fatigue and the microbiota and mycobiota.MethodsAfter obtaining approval from the ethics board, we collected 100 BALF samples from 10 patients with silicosis. Demographic data, clinical information, and blood test results were also collected from each patient. The characteristics of the microbiota and mycobiota were defined using next-generation sequencing. However, no non-silicosis referent group was examined, which was a major limitation of this study.ResultsOur analysis indicated that subsampling from different rounds of BALF did not affect the alpha- and beta-diversities of microbial and fungal communities when the centrifuged BALF sediment was sufficient for DNA extraction. In contrast, fatigue status significantly influenced the beta-diversity of microbes and fungi (Principal Coordinates Analysis, P = 0.001; P = 0.002). The abundance of Vibrio alone could distinguish silicosis patients with fatigue from those without fatigue (area under the curve = 0.938, 95% confidence interval [CI] 0.870-1.000). Significant correlations were found between Vibrio and haemoglobin levels (P < 0.001, ρ = -0.64).ConclusionsSampling in different rounds of BALF showed minimal effect on BALF microbial and fungal diversities; the first round of BALF collection was recommended for microbial and fungal analyses for convenience. In addition, Vibrio may be a potential biomarker for silicosis fatigue screening.
| S-EPMC10332100 | biostudies-literature