Bronchoalveolar Lavage Research Articles

One-Step Soft Agar Enrichment and Isolation of Human Lung Bacteria Inhibiting the Germination of Aspergillus fumigatus Conidia

Fungi of the genus Aspergillus are widespread in the environment, where they produce large quantities of airborne conidia. Inhalation of Aspergillus spp. conidia in immunocompromised individuals can cause a wide spectrum of diseases, ranging from hypersensitivity responses to lethal invasive infections. Upon deposition in the lung epithelial surface, conidia encounter and interact with complex microbial communities that constitute the lung microbiota. The lung microbiota has been suggested to influence the establishment and growth of Aspergillus spp. in the human airways. However, the mechanisms underlying this interaction have not yet been sufficiently investigated. In this study, we aimed to enrich and isolate bacterial strains capable of inhibiting the germination and growth of A. fumigatus conidia from bronchoalveolar lavage fluid (BALF) samples of lung transplant recipients using a novel enrichment method. This method is based on a soft agar overlay plate assay in which bacteria are directly in contact with conidia, allowing inhibition to be readily observed during enrichment. We isolated a total of five clonal bacterial strains with identical genotypic fingerprints, as shown by random amplified polymorphic DNA PCR (RAPD–PCR). All strains were identified as Pseudomonas aeruginosa (strains b1–b5). The strains were able to inhibit the germination and growth of Aspergillus fumigatus in a soft agar confrontation assay, as well as in a germination multiplate assay. Moreover, when compared with ten P. aeruginosa strains isolated from expectoration through standard methods, no significant differences in inhibitory potential were observed. Additionally, we showed inhibition of A. fumigatus growth on Calu-3 cell culture monolayers. However, the isolated P. aeruginosa strains were shown to cause significant damage to the cell monolayers. Overall, although P. aeruginosa is a known opportunistic lung pathogen and antagonist of A. fumigatus, we validated this novel one-step enrichment approach for the isolation of bacterial strains antagonistic to A. fumigatus from BALF samples as a proof-of-concept. This opens up a new venue for the targeted enrichment of antagonistic bacterial strains against specific fungal pathogens.

Exploring the Impact of Molecular Hydrogen Therapy on Oxidative Stress in Bleomycin-Induced Idiopathic Pulmonary Fibrosis: A Research Protocol

Introduction: Idiopathic Pulmonary Fibrosis (IPF) is an intensifying respiratory disorder triggered by bleomycin in cancer patients, characterized by its lack of a definitive remedy. Bleomycin induces mitochondrial leakage and elevates Reactive Oxygen Species (ROS) in the pulmonary cavity, causing a reduction in glutathione (GSH), a vital lung antioxidant. This reduction in GSH levels, coupled with ROS's inactivation of the tumor suppressor gene p53, increases the chance of developing fibrosis. Although no antioxidant treatments that currently target these effects, Molecular Hydrogen Therapy (MHT) has proven effective in mitigating ROS in diseases like Chronic Obstructive Pulmonary Disease (COPD). This study aims to explore the impact of bleomycin-induced IPF on ROS and p53, investigating MHT as a potential treatment to alleviate oxidative stress in fibrosis. Methods: The experimental design includes four groups of mice, two receiving bleomycin injections and two receiving a control buffer solution. Within each subgroup, one group will undergo MHT, while the other will receive sterile air. Bronchoalveolar lavage will be used to measure GSH levels before and after MHT, and RNA sequencing will monitor p53 activity in all treatment and control groups. Results: Anticipated outcomes include elevated GSH levels in the MHT-treated bleomycin group, indicating an antioxidative effect. Consequently, improved p53 activity is expected compared to controls. Discussion: Anticipated results, based on existing literature, suggest that MHT enhances antioxidant defenses and modulates p53 activity, thereby reducing oxidative damage and fibrotic remodeling. These findings highlight the potential of MHT as a therapeutic intervention for IPF, with broader implications for managing other oxidative stress-mediated lung diseases. However, further research, including preclinical studies and clinical trials, is needed to validate these anticipated findings and ensure the long-term safety and efficacy of MHT in clinical practice. Conclusion: The study aims to replicate findings in human populations and proposes a paradigm shift in treating such diseases by addressing the root cause, ROS.

The clinical application of metagenomic next-generation sequencing in immunocompromised patients with severe respiratory infections in the ICU.

Early targeted antibiotic therapy is crucial for improving the prognosis of immunocompromised patients with severe respiratory infections (SRIs) in the intensive care unit (ICU). Metagenomic next-generation sequencing (mNGS) has shown significant value in pathogen detection, but research on lower respiratory tract microorganisms remains limited. This study enrolled 234 patients with SRIs in the ICU, and individuals were categorized into immunocompromised and immunocompetent groups. We compared the diagnostic performance of mNGS using bronchoalveolar lavage fluid (BALF) with conventional microbiological tests (CMTs) and analyzed the value of mNGS in immunocompromised patients with SRIs in the ICU. Among all patients, the pathogenic microorganism detection rate of mNGS was higher than that of CMTs (94.02% vs 66.67%, P < 0.05), both in the immunocompromised group (95.0% vs 58.75%, P < 0.05) and the immunocompetent group (93.51% vs 71.43%, P < 0.05). mNGS detected more pathogens than CMTs did (167 vs 51), identifying 116 organisms that were missed by CMTs. The proportion of antibiotic regimen adjustments based on mNGS results was significantly higher compared to CMTs in both the immunocompromised (70.00% vs 17.50%, P < 0.05) and immunocompetent groups (48.70% vs 15.58%, P < 0.05). In the immunocompromised group, patients who had their antibiotic treatment adjusted on mNGS results had improved prognosis, with significantly lower ICU mortality (8.93% vs 50%, P < 0.05) and 28-day mortality rates (30.36% vs 68.75%, P < 0.05) than CMTs. In the immunocompetent group, no statistically significant differences were observed in ICU mortality or 28-day mortality (20.00% vs 33.33%, P > 0.05; 42.67% vs 45.83%, P > 0.05). mNGS shows significant value in detecting pathogens in immunocompromised patients with SRIs in ICU. For immunocompromised patients who respond poorly to empirical treatment, mNGS can provide an etiological basis, helping adjust antibiotic regimens more precisely and thereby improving patient prognosis.

Single-cell landscape of bronchoalveolar immune cells in patients with immune checkpoint inhibitor-related pneumonitis

The pathophysiology of immune checkpoint inhibitor-related pneumonitis remains incompletely understood. We conducted single-cell and T-cell receptor transcriptomic sequencing on the bronchoalveolar lavage fluid from five patients with grade ≥2 immune checkpoint inhibitor-related pneumonitis. Our analyses revealed a prominent enrichment of T cells in the bronchoalveolar lavage fluid of patients with immune checkpoint inhibitor-related pneumonitis. Within the CD4 + T cell subset, Tfh-like T cells were highly enriched and exhibited signatures associated with inflammation and clonal expansion. Regulatory T cells were also enriched and displayed enhanced inhibitory functions. Within the CD8 + T-cell subset, effector memory/tissue-resident memory T cells with an elevated cytotoxic phenotype were highly infiltrated. Among myeloid cells, alveolar macrophages were depleted, while pro-inflammatory intermediate monocytes were elevated. Dendritic cells demonstrated enhanced antigen presentation capabilities. Cytokines CXCR4, CXCL13, TNF-α, IFN-α, IFN-γ, and TWEAK were elevated. Through a comprehensive single-cell analysis, we depicted the landscape of immune checkpoint inhibitor-related pneumonitis.

Diagnostic Value of Targeted Next-generation Sequencing in Pulmonary Mycobacterial Infections.

This study aimed to explore the diagnostic value of novel technique-targeted next-generation sequencing (tNGS) of bronchoalveolar lavage fluid (BALF) in pulmonary mycobacterial infections. This retrospective study was conducted on patients who underwent bronchoscopy and tNGS, smear microscopy, and mycobacterial culture of BALF. Patients with positive Mycobacterium tuberculosis (MTB) culture or GeneXpert results were classified into the tuberculosis case group. Those diagnosed with nontuberculous mycobacteria (NTM)-pulmonary disease (NTM-PD) composed the case group of NTM-PD patients. The control group comprised patients without tuberculosis or NTM-PD. Sensitivity, specificity, and receiver operating characteristic (ROC) curves were used to evaluate the diagnostic performance. For tuberculosis patients with positive mycobacterial culture results, the areas under the ROC curves (AUCs) for tNGS, GeneXpert, and smear microscopy were 0.975 (95% CI: 0.935, 1.000), 0.925 (95% CI: 0.859, 0.991), and 0.675 (95% CI: 0.563, 0.787), respectively. For tuberculosis patients with positive GeneXpert results, the AUCs of tNGS, culture, and smear microscopy were 0.970 (95% CI: 0.931, 1.000), 0.850 (95% CI: 0.770, 0.930), and 0.680 (95% CI: 0.579, 0.781), respectively. For NTM-PD, the AUCs of tNGS, culture, and smear-positive but GeneXpert-negative results were 0.987 (95% CI: 0.967, 1.000), 0.750 (95% CI: 0.622, 0.878), and 0.615 (95% CI: 0.479, 0.752), respectively. The sensitivity and specificity of tNGS in NTM-PD patients were 100% and 97.5%, respectively. tNGS demonstrated superior diagnostic efficacy in mycobacterial infection, indicating its potential for clinical application.

Screening the effective components of Lysionotus pauciflorus Maxim. on the treatment of LPS induced acute lung injury mice by integrated UHPLC-Q-TOF-MS/MS and network pharmacology

Ethnopharmacological relevanceAcute lung injury (ALI) is an inflammatory reaction produced through various injury-causing factors acting on the lungs in a direct or indirect way, with a high morbidity and mortality rate. A review of clinical experience has revealed that Lysionotus pauciflorus Maxim (LP) has a significant therapeutic effect on ALI. However, the comprehensive effective components of LP are uncertain, and the mechanisms, especially the potential therapeutic target for anti-ALI, are still unknown. Aims of the studyIn vitro and in vivo validation of the pharmacodynamics of LP in the treatment of ALI and exploration of its potential mechanism of action based on network pharmacology, molecular docking and experimental validation. Materials and methodsUltra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was employed to identify the ingredients of LP extracts. The potential bioactive ingredients, key targets and signalling pathways were identified by network pharmacology, based on the results of the mass spectrometry analysis. Subsequently, molecular docking was performed on the screened core components and key targets to calculate their molecular binding energies and binding potentials, and to explore the mutual binding modes of small-molecule ligands and large-molecule proteins. Finally, lipopolysaccharide (LPS)-induced RAW264.7 cell model and ALI mice model were used to validate the therapeutic effects and potential mechanism of LP extract towards ALI. ResultsFrom the mass spectrometry results of LP extracts, a total of 89 chemical components were identified, including 46 phenylethanol glycosides, 26 flavonoids, 9 organic acids and their derivatives and 8 other compounds. And furthermore 39 core active components were screened by network pharmacology. The top 10 core components (4 phenylethanol glycosides, 6 flavonoids) have been screened in the composition -target-disease network, and 37 core targets related to LP efficacy were obtained by fitting PPI network analysis. 10 signalling pathways and their targets associated with LP treatment of ALI were obtained by GO/KEGG analysis, indicating that LP could regulate TLR4 and NF-κB signalling pathways through 4 key targets, namely NFKB1, RELA, TLR4 and TNF. The results of the molecular docking procedure indicated a strong affinity, with the binding energies between each component and the target site being less than −6 kcal/mol. The binding modes included Hydrogen Bonds, Pi-Pi interaction, Hydrophobic Interactions, Salt Bridges, Pi-cation interactions. These observations were subsequently validated in vitro and in vivo experiments. The outcomes of in vitro and in vivo experiments demonstrated that LP was effective in reducing the infiltration of inflammatory bacteria in lung tissues and attenuated the expression of pro-inflammatory cytokines in LPS-stimulated mice bronchoalveolar lavage fluid (BALF) and RAW264.7 cells. Furthermore, LP inhibited the expression and phosphorylation of TLR4 protein and NF-κB protein, thus playing a role in the prevention of ALI. ConclusionsIn this study, mass spectrometry analysis was combined with biomolecular networks to initially elucidate the potential of LP to treat ALI by modulating the TLR4/NF-κB pathway. This offers a definitive experimental basis for the development of new LP drugs.

9332 Breathless Struggle: Unraveling The Interplay Between DKA And Acute Respiratory Distress Syndrome

Abstract Disclosure: S. Zahra: None. R. Subramani: None. M. Abbas: None. K.Y. Hasan: None. F. Manas: None. C. Bajracharya: None. Introduction: Diabetic Ketoacidosis (DKA) is a life threatening emergency with mortality rate of 0.15%-0.30%. Acute Respiratory Distress Syndrome (ARDS) is a rare complication of DKA which presents as a sudden onset of dyspnea and progressive hypoxia. It is an inflammatory process that damages the alveolar-capillary membrane leading to the fluid leakage into the alveolar space. The incidence of ARDS in DKA remains unknown. It is more commonly seen in adolescents and young adults. The exact pathogenesis of ARDS in DKA is currently unknown. Some proposed mechanisms are that a severe metabolic acidosis in DKA along with increased cytokines production due to long standing hyperglycemia leads to increased capillary membrane permeability allowing fluid to accumulate in the alveolar space. TNF-a and IL-6 along with other pro-inflammatory cytokines are known to cause endothelial damage and increase pulmonary permeability leading to pulmonary edema. However most DKA patients with severe metabolic acidosis do not develop ARDS. Case Presentation:Case 1: A 19 year old male with uncontrolled type I diabetes mellitus presented with altered mental status. On examination, he was tachycardic and tachypneic with dry mucous membranes. Lab workup revealed leukocytosis, severe hyperglycemia, and severe anion gap metabolic acidosis with beta hydroxybutyrate level of 14.20 mmol/L (normal: 0.02-0.27 mmol/L). He was diagnosed with DKA and started on intravenous fluids, and insulin as per institutional DKA protocol. Approximately 12 hours post-admission, the patient experienced sudden-onset dyspnea with deteriorating hypoxia, necessitating emergent intubation and mechanical ventilation. Chest radiography displayed bilateral patchy ground-glass opacifications consistent with acute respiratory distress syndrome (ARDS). Despite the administration of 8 liters of intravenous fluids, the patient exhibited a negative net fluid balance of 2.3 liters, implicating fluid shift. Further investigations, including bronchoscopy and bronchoalveolar lavage, revealed an unobstructed airway, minimal secretions, and no evidence of hemorrhage or infection. Comprehensive infectious work-ups yielded negative results as well, reinforcing the diagnosis of DKA as the underlying cause of ARDS. Conclusion: Acute Respiratory Distress Syndrome is a rare but potentially fatal complication of Diabetic Ketoacidosis. Early recognition of this serious pulmonary complication of DKA and its prompt management is essential to prevent respiratory failure and mortality. Presentation: 6/1/2024

Microplastics, as a risk factor in the development of interstitial lung disease- a preliminary study

Microplastic (MPs) pollution is a global concern that affects all living organisms, yet research on MP-related disorders in humans, including incidence and symptoms, remains limited. In this study, the presence, composition, and characterization of MPs in bronchoalveolar lavage (BAL), which reflects lung tissue, and blood were examined. Fiberoptic bronchoscopy was performed to collect BAL samples from patients suspected of having interstitial lung disease (ILD) as well as from a control group. MPs were identified and measured using μ-Raman techniques.In BAL samples, the most common MPs color observed was grey/white, with sizes ranging from 4.19 to 792.00 μm. The particle shapes and polymer types identified included polyamide (PA), polyester (PET), polyvinyl chloride (PVC), and polyurethanes (PU). For blood samples, MPs were predominantly grey/white and blue, with sizes ranging from 13.14 to 20. 29 μm. The identified polymers in blood samples included polyamide (PA) and polyethylene (PE). MPs were detected in 10 out of 18 patients (55%) suspected of having ILD, with most of these patients presenting with the fibrotic type of the disease. In the control group, two patients whose BAL samples were positive for MPs were found to have chronic lung disease.This study is the first to explore the relationship between interstitial lung disease (ILD) and microplastics (MPs), revealing a tendency for the presence of MPs in the bronchoalveolar lavage (BAL) of ILD patients, particularly those with a fibrotic phenotype. Further research is needed to determine the cumulative effects of MPs on human health, especially concerning the respiratory system, which is highly exposed to environmental pollutants.

Profibrotic monocyte-derived alveolar macrophages are expanded in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19

Monocyte-derived alveolar macrophages drive lung injury and fibrosis in murine models and are associated with pulmonary fibrosis in humans. Monocyte-derived alveolar macrophages have been suggested to develop a phenotype that promotes lung repair as injury resolves. We compared single-cell and cytokine profiling of the alveolar space in a cohort of 35 patients with post-acute sequelae of COVID-19 who had persistent respiratory symptoms and abnormalities on a computed tomography scan of the chest that subsequently improved or progressed. The abundance of monocyte-derived alveolar macrophages, their gene expression programs, and the level of the monocyte chemokine CCL2 in bronchoalveolar lavage fluid positively associated with the severity of radiographic fibrosis. Monocyte-derived alveolar macrophages from patients with resolving or progressive fibrosis expressed the same set of profibrotic genes. Our findings argue against a distinct reparative phenotype in monocyte-derived alveolar macrophages, highlighting their utility as a biomarker of failed lung repair and a potential target for therapy.

A quantitative proteomic approach to evaluate the efficacy of carnosine in a murine model of chronic obstructive pulmonary disease (COPD)

The aim of the work was to study a dose-dependent effect of inhaled carnosine (10, 50 or 100 mg/kg/day) in mice exposed to cigarette smoke as a model of chronic obstructive pulmonary disease (COPD). A dose-dependent loading of the dipeptide in lung tissue and bronchoalveolar lavage (BAL) was firstly demonstrated by LC-ESI-MS analysis. Cigarette smoke exposure induced a significant lung inflammation and oxidative stress in mice which was dose-dependently reduced by carnosine. Inflammation was firstly evaluated by measuring the cytokines content in the BAL. All the measured cytokines were found significantly higher in the smoke group in respect to control, although the data are affected by a significant variability. Carnosine was found effective only at the highest dose tested and significantly only for keratinocyte-derived cytokine (KC). Due to the high variability of cytokines, a quantitative proteomic approach to better understand the functional effect of carnosine and its molecular mechanisms was used. Proteomic data clearly indicate that smoke exposure had a great impact on lung tissue with 692 proteins differentially expressed above a threshold of 1.5-fold. Protein network analysis identified the activation of some pathways characteristic of COPD, including inflammatory response, fibrosis, induction of immune system by infiltration and migration of leukocyte pathways, altered pathway of calcium metabolism and oxidative stress. Carnosine at the tested dose of 100 mg/kg was found effective in reverting all the pathways evoked by smoke. Only a partial reverse of the dysregulated proteins was evident at low- and mid-tested doses, although, for some specific proteins, indicating an overall dose-dependent effect. Regarding the molecular mechanisms involved, we found that carnosine upregulated some key enzymes related to Nrf2 activation and in particular glutathione peroxidase, reductase, transferase, SOD, thioredoxins, and carbonyl reductase. Such mechanism would explain the antioxidant and anti-inflammatory effects of the dipeptide.

Cordyceps militaris Grown on Germinated Rhynchosia nulubilis (GRC) Encapsulated in Chitosan Nanoparticle (GCN) Suppresses Particulate Matter (PM)-Induced Lung Inflammation in Mice.

Cordyceps militaris grown on germinated Rhynchosia nulubilis (GRC) exerts various biological effects, including anti-allergic, anti-inflammatory, and immune-regulatory effects. In this study, we investigated the anti-inflammatory effects of GRC encapsulated in chitosan nanoparticles (CN) against particulate matter (PM)-induced lung inflammation. Optimal CN (CN6) (CHI: TPP w/w ratio of 4:1; TPP pH 2) exhibited a zeta potential of +22.77 mV, suitable for GRC encapsulation. At different GRC concentrations, higher levels (60 and 120 mg/mL) led to increased negative zeta potential, enhancing stability. The optimal GRC concentration for maximum entrapment (31.4 ± 1.35%) and loading efficiency (7.6 ± 0.33%) of GRC encapsulated in CN (GCN) was 8 mg/mL with a diameter of 146.1 ± 54 nm and zeta potential of +30.68. In vivo studies revealed that administering 300 mg/kg of GCN significantly decreased the infiltration of macrophages and T cells in the lung tissues of PM-treated mice, as shown by immunohistochemical analysis of CD4 and F4/80 markers. Additionally, GCN ameliorated PM-induced lung tissue damage, inflammatory cell infiltration, and alveolar septal hypertrophy. GCN also decreased total cells and neutrophils, showing notable anti-inflammatory effects in the bronchoalveolar lavage fluid (BALF) from PM-exposed mice, compared to GRC. Next the anti-inflammatory properties of GCN were further explored in PM- and LPS-exposed RAW264.7 cells; it significantly reduced PM- and LPS-induced cell death, NO production, and levels of inflammatory cytokine mRNAs (IL-1β, IL-6, and COX-2). GCN also suppressed NF-κB/MAPK signaling pathways by reducing levels of p-NF-κB, p-ERK, and p-c-Jun proteins, indicating its potential in managing PM-related inflammatory lung disease. Furthermore, GCN significantly reduced PM- and LPS-induced ROS production. The enhanced bioavailability of GRC components was demonstrated by an increase in fluorescence intensity in the intestinal absorption study using FITC-GCN. Our data indicated that GCN exhibited enhanced bioavailability and potent anti-inflammatory and antioxidant effects in cells and in vivo, making it a promising candidate for mitigating PM-induced lung inflammation and oxidative stress.

Luteolin alleviates airway remodeling in asthma by inhibiting the epithelial-mesenchymal transition via β-catenin regulation

BackgroundAsthma is a prevalent long-term inflammatory condition that causes airway inflammation and remodeling. Increasing evidence indicates that epithelial-mesenchymal transition (EMT) holds a prominent implication in airway reconstruction in patients with asthma. Flavonoids obtained from Chinese Materia Medica (CMM), such as Luteolin (Lut), exhibit various beneficial effects in various asthma models. Lut has been shown to mitigate various asthma symptoms, including airway inflammation, hyperresponsiveness, bronchoconstriction, excessive mucus production, pulmonary autophagy, and neutrophilic asthma. However, whether flavonoids can suppress EMT-associated airway remodeling in asthma and the fundamental mechanisms involved remain unclear, with no studies specifically addressing Lut in this context. PurposeTo evaluate the inhibition of airway remodeling in asthma by Lut and its potential mechanisms, while examining the significance of β-catenin in this process through cellular and animal studies. MethodsA BEAS-2B cell model stimulated by lipopolysaccharide (LPS) was established in vitro. Wound closure and Transwell assays were utilized to assess the cellular migratory ability. EMT- and fibrosis-related markers in LPS-stimulated cells were evaluated using RT-qPCR and western blotting. The status of the β-catenin/E-cadherin and β-catenin destruction complexes was evaluated using western blotting, immunofluorescence (IF) staining, and co-immunoprecipitation (Co-IP) analysis. The regulatory function of Lut in β-catenin-dependent EMT was further validated by β-catenin overexpression with adenovirus transduction and siRNA-mediated knockdown of β-catenin. Moreover, the counts of different types of bronchoalveolar lavage fluid (BALF) inflammatory cells from mice with asthma induced by ovalbumin (OVA) were evaluated in vivo using Congo red staining. Hematoxylin and eosin (H&E), Masson's trichrome, and periodic acid-Schiff (PAS) staining were used to evaluate collagen deposition, mucus production, and inflammation in murine lung tissues. Western blotting and immunohistochemistry (IHC) assays were used to assess EMT- and fibrosis-related markers in the lung tissues in vivo. ResultSix naturally derived flavonoids, including Lut, attenuated cell migration and prevented EMT in LPS-treated BEAS-2B cells. Moreover, Lut suppressed TGF-β1, MMP-9, fibronectin (FN), and α-smooth muscle actin (α-SMA) levels in LPS-stimulated BEAS-2B cells. Additionally, Lut downregulated the levels of β-catenin by modulating the β-catenin/E-cadherin and β-catenin destruction complexes, highlighting the pivotal role of β-catenin in EMT inhibition by Lut in LPS-stimulated BEAS-2B cells. Furthermore, Lut suppressed airway inflammation and attenuated EMT-associated airway remodeling through β-catenin blockade in OVA-induced asthmatic mice. The bronchial wall thickness notably reduced from 37.24 ± 4.00 μm in the asthmatic model group to 30.06 ± 4.40 μm in the Lut low-dose group and 24.69 ± 2.87 μm in the Lut high-dose group. ConclusionAccording to our current understanding, this research is the first to reveal that Lut diminishes airway remodeling in asthma by inhibiting EMT via β-catenin regulation, thereby filling a research gap concerning Lut and flavonoids. These results provide a theoretical basis for treating asthma with anti-asthmatic CMM, as well as a candidate and complementary therapeutic approach to treat asthma.

Rothia mucilaginosa Pneumonia in an Immunocompromised Patient

Abstract Rothia mucilaginosa is a gram-positive coccus recognized as an opportunistic pathogen capable of causing pneumonia in immunocompromised patients (Antimicrob Agents Chemother. 1995;39:268–270). The organism is often resistant to fluoroquinolones and trimethoprim/sulfamethoxazole, agents routinely used as anti-infective prophylaxis in immunocompromised individuals (Inf Dis Clin Pract. 2020;28:361–365; Infect Dis Now. 2021;51:228–235). Plasma next-generation sequencing (NGS) is increasing in clinical use to aid in diagnosis of infections in immunocompromised patients (Infect Dis (Lond). 2015;47:125–1299). A patient with advanced multiple myeloma was admitted with pneumonia. Plasma NGS initially suggested R. mucilaginosa, which was confirmed by cultures of bronchoalveolar lavage (BAL) fluid. Treatment with eravacycline led to clinical and radiographic resolution of pneumonia.

Integrating network pharmacology and transcriptomics to study the potential mechanism of Jingzhi Niuhuang Jiedu tablet in rats with accumulation of heat in the lungs and stomach

Ethnopharmacological relevanceAccumulation of heat in the lungs and stomach (AHLS) is an important syndrome within the realm of traditional Chinese medicine (TCM). It is the fundamental reason behind numerous illnesses, including mouth ulcers, dermatological conditions, acne, and pharyngitis. Jingzhi Niuhuang Jiedu tablet (JN) serves as the representative prescription for treatment of AHLS clinically. However, the effective components and mechanism of JN's impact on AHLS remain unexplored. Aim of the studyThe objective of this research was to analyze the effective components of JN and investigate the therapeutic effect and potential mechanism of JN on AHLS. Materials and methodsThe effective compounds of JN extract were analyzed and identified using UHPLC-Q-Exactive/HRMS. Utilizing network pharmacology to investigate JN's multi-target, multi-pathway process in treating AHLS. Subsequently, anti-inflammatory activities of JN extract were evaluated in the RAW264.7 cells stimulated by lipopolysaccharide (LPS). In addition, a rat AHLS model induced by LPS and dried ginger was established. Pathological changes in rat lung and stomach tissues observed by HE staining and Masson's trichrome staining. Additionally, the expression of TNF-α, IL-6, and IL-1β in bronchoalveolar lavage fluid (BALF) was identified through the ELISA assay. For a deeper understanding of how JN might affect AHLS, transcriptomics was utilized to examine differential genes and their underlying mechanisms. Concurrently, techniques like quantitative polymerase chain reaction (q-PCR), immunofluorescence, and western blotting (WB) were employed to confirm various mRNA and protein expression, including Il17ra, Il17re, IL-17A, IL-1β, IL-6, PPARγ, PGC1-α and UCP1. ResultsWe identified 178 potential effective components in the JN extract. Network pharmacology analysis showed that the 144 components in JN act on 200 key targets for the treatment of AHLS by suppressing inflammation, regulating energy metabolism, and gastric function. In addition, JN suppressed the LPS-stimulated generation of NO, TNF-α, IL-1β, and IL-6 in RAW264.7 cells. And JN treatment effectively alleviated lung and stomach injury and reduced inflammation in rats. Analysis of RNA-seq from lung tissues revealed JN's substantial control over crucial genes in the IL-17 signaling pathway, including Il1b and Il17ra. Likewise, RNA sequencing of stomach tissues revealed that JN markedly decreased crucial genes in the Thermogenesis pathway, including Ppargc1a and Ppara. Additional experimental findings confirmed that treatment with JN significantly reduced the expression levels of mRNA (Il17ra, Il17re, Il1b, Ppargc1a and Ucp1), and the expression levels of protein (IL-17A, IL-1β, IL-6, PPARγ, PGC1-α and UCP1). ConclusionThis study not only analyzes the effective components of JN but also reveals that JN could effectively ameliorate AHLS by inhibiting IL-17 signaling pathway and Thermogenesis pathway, which provides evidence for subsequent clinical studies and drug development.

B-198 Rapid Molecular Detection of Respiratory Infections in Brazilian Hospitals and Outpatient Units: Insights from the Diagnostic Medicine and Health Network

Abstract Background Diagnosing agents causing respiratory infections remains a global challenge due to the numerous coexisting agents and similar symptoms. This study evaluates the epidemiology of viral and bacterial agents responsible for major respiratory infections through molecular detection in patients across various Brazilian hospital units served by a clinical diagnostic and health laboratory. Understanding the distribution and prevalence of these pathogens is crucial for effective diagnosis, treatment, and prevention strategies in the face of evolving epidemiological landscapes. Methods Utilizing an internal database, we conducted a comprehensive analysis of tests performed on the FilmArray® system. This system employs multiplex PCR technology, capable of simultaneously identifying 21 respiratory agents in a single sample obtained from nasopharyngeal and bronchial lavage specimens collected from diverse geographical regions across Brazil. The data for the examination of patient samples with respect to gender, age group, geographical regions, and the prevalence of various agents between January 2021 and April 2023 were retrieved from the database and analyzed. To ensure privacy, sensitive and individual data from all patients were meticulously preserved throughout the study. Results The analysis encompassed a diverse sample of 477 patients, originating from 26 cities across 11 Brazilian states, including Goiás, Minas Gerais, Mato Grosso, Pará, Paraná, Rio de Janeiro, São Paulo, Tocantins, Amazonas, Bahia, and Distrito Federal. The distribution of patients was nearly split between hospital settings (39.8%) and outpatient units, with hospital patients predominantly from departments such as internal medicine, cardiology, and nephrology. In contrast, outpatient samples were primarily requested by specialists in pulmonology and infectious diseases. The demographic profile revealed an average age of 36 years, with a majority male representation (54%). However, a notable shift occurred in the first four months of 2023, where females constituted 60.8% of the sample. The overall test positivity rate stood at 42.3%, with a significant detection of 71% (15 out of 21) of the surveyed respiratory agents at some point during the study period. The most frequently identified pathogens were Rhinovirus, Enterovirus, and SARS-CoV-2, collectively accounting for 55.1% of the positive findings. Additionally, mixed infections were observed in 29 (6%) of the patients, with combinations such as SARS-CoV-2 and Rhinovirus, SARS-CoV-2 and H3N2, and SARS-CoV-2 and RSV, among others, highlighting the complexity of respiratory infections. Conclusions The findings from this study underscore the critical role of advanced molecular diagnostics in the Brazilian clinical-laboratory landscape. The utilization of multiplex PCR technology has proven to be a pivotal tool in accurately identifying a wide array of respiratory pathogens, thereby enhancing the precision of diagnosis and treatment strategies. The detection of a high prevalence of agents such as Rhinovirus, Enterovirus, and SARS-CoV-2, along with the identification of mixed infections, highlights the complexity of respiratory infections and the necessity for robust diagnostic tools. This study not only contributes to the understanding of the epidemiology of respiratory infections in Brazil but also emphasizes the importance of continuous advancements in molecular diagnostics for better patient care and public health management.

Identification and Validation of a Threshold for Early Posttransplant Bronchoalveolar Fluid Hyaluronan that Distinguishes Lung Recipients at Risk for CLAD

BackgroundFew tools exist for early identification of patients at risk for chronic lung allograft dysfunction (CLAD). We previously showed hyaluronan (HA), a matrix molecule that regulates lung inflammation and fibrosis, accumulates in bronchoalveolar lavage fluid (BALF) and blood in CLAD. We aimed to determine if early posttransplant HA elevations inform CLAD risk. MethodsHA was quantified in 3080 BALF and 1323 blood samples collected over the first posttransplant year in 743 adult lung recipients at 5 centers. The relationship between BALF or blood HA and CLAD was assessed using Cox models with a time-dependent binary covariate for “elevated” HA. Potential thresholds for elevated HA were examined using a grid search between the 50th and 85th percentile. The optimal threshold was identified using fit statistics, and the association between the selected threshold and CLAD was internally validated through iterative resampling. A multivariable Cox model using the selected threshold was performed to evaluate the association of elevated HA with CLAD considering other factors that may influence CLAD risk. ResultsBALF HA levels >19.1ng/mL (65th percentile), had the largest hazard ratio for CLAD (HR 1.70, 95% CI 1.25–1.31; p<0.001), optimized fit statistics, and demonstrated robust reproducibility. In a multivariable model, the occurrence of BALF HA >19.1 ng/mL in the first posttransplant year conferred a 66% increase in the hazards for CLAD (adjusted HR 1.66, 95% CI 1.19–2.32; p=0.003). Blood HA was not significantly associated with CLAD. ConclusionsWe identified and validated a precise threshold for BALF HA in the first posttransplant year that distinguishes patients at increased CLAD risk.

A Randomized Control Trial Comparing the Yield of Bronchoalveolar Lavage Using Three Different Techniques in Patients Undergoing Flexible Bronchoscopy (BAL-3T).

Three techniques have been described for aspirating the bronchoalveolar lavage (BAL) fluid, namely the wall mount suction (WMS), manual suction (MS), and manual suction with tubing (MST). However, there is no direct comparison among the 3 methods. We randomized patients undergoing flexible bronchoscopy and BAL in a 1:1:1 ratio to one of the 3 arms. The primary outcome was to compare the optimal yield, defined as at least 30% return of volume instilled and <5% bronchial cells. The key secondary outcomes were the percentage of volume and total amount (in millimeters) return of BAL, as well as complications (hypoxemia, airway bleeding, and others). We randomized 942 patients [MST (n = 314), MS (n = 314), WMS (n = 314)]. The mean age of the study population [58.7% (n = 553) males] was 46.9 years. The most common indication for BAL was suspected pulmonary infection. Right upper lobes and middle lobes were the commonest sampled lobes. The optimal yield was similar in all the groups [MST (35.6%) vs MS (42.2%) vs WMS (36.5%); P = 0.27]. A significantly higher proportion of patients had BALF return >30% (P = 0.005) in the WMS (54.2%) and MS (54%) than in the MST arm (42.9%). The absolute and the percentage volume of BALF was also higher in WMS and MS than in the MST arm. There was no difference in the complication rate or other secondary outcomes across the groups. We found no difference in the optimal yield of BAL or complications using any one of the 3 methods for BAL fluid retrieval.

A Differential Protein Study on Bronchoalveolar Lavage Fluid at Different Stages of Silicosis.

In this study, by comparing the difference in protein expression in bronchoalveolar lavage fluid between silicosis patients in different stages and healthy controls, the pathogenesis of pneumoconiosis was discussed, and a new idea for the prevention and treatment of pneumoconiosis was provided. The lung lavage fluid was pretreated by 10 K ultrafiltration tube, Agilent 1100 conventional liquid phase separation, strong cation exchange column (SCX) HPLC pre-separation, and C18 reverse phase chromatography desalting purification, and protein was labeled with isotope. GO, KEGG pathway, and PPI analysis of differential proteins were conducted by bioinformatics, and protein types and corresponding signal pathways were obtained. Thermo Q-Exactive mass spectrometry identified 943 proteins. T-test analysis was used to evaluate the different significance of the results, and the different protein of each group was obtained by screening with the Ratio≥1.2 or Ratio≤0.83 and P<0.05. We found that there are 16 kinds of protein throughout the process of silicosis. There are different expressions of protein in stages Ⅲ/control, stages Ⅱ/control, stage Ⅰ/control, stages Ⅲ/ stages Ⅱ, stages Ⅲ/ stage Ⅰ and stages Ⅱ/ stage Ⅰ groups. The results of ontology enrichment analysis of total differential protein genes show that KEGG pathway enrichment analysis of differential protein suggested that there were nine pathways related to silicosis. The main biological changes in the early stage of silicosis are glycolysis or gluconeogenesis, autoimmunity, carbon metabolism, phagocytosis, etc., and microfibril-associated glycoprotein 4 may be involved in the early stage of silicosis. The main biological changes in the late stage of silicosis are autoimmunity, intercellular adhesion, etc. Calcium hippocampus binding protein may participate in the biological changes in the late stage of silicosis. It provides a new idea to understand the pathogenesis of silicosis and also raises new questions for follow-up research.

Experimental and proteomics evidence revealed the protective mechanisms of ShemaZhichuan Liquid in attenuating neutrophilic asthma

BackgroundNeutrophilic asthma (NA) is one of the most important phenotypes of non-Th2 asthma and is often insensitive to glucocorticoid therapy, making current treatment difficult. Shemazhichuan Liquid (SMZCL), a Chinese medicine compound preparation, has unique advantages in the treatment of asthma. However, the underlying mechanisms of SMZCL in treating NA are not fully understood. PurposeThe efficacy and underlying mechanisms of SMZCL on NA were investigated by TMT-labeled quantitative proteomics analysis and in vivo and in vitro experiments. MethodsNA mouse model was constructed by OVA/CFA sensitization followed by a 10-day challenge with 5% OVA. Lung histopathology, leukocyte counts and cell sorting counts, inflammatory cytokines levels, as well as expression of autophagy markers were then assessed. The specific pathways and proteins of SMZCL for treating NA were further illustrated through TMT-based quantitative proteomics. In addition, RAW264.7 cells were induced by LPS to further explore the mechanism of the main active ingredient of SMZCL on autophagy pathway. ResultsIn vivo, SMZCL contributed to attenuating airway inflammation and collagen disposition, markedly reduced the number of leukocytes, especially neutrophils in bronchoalveolar lavage fluid (BALF), as well as decreased IgE and inflammatory cytokine levels (TNF-α, IL-1β, IL-6 and IL-8) in BALF and serum. Besides, SMZCL elevated the levels of LC3 and ATG5 while inhibiting the expression of p62 and mTOR. Mzb1 and Rab3ip were identified as the critical overlapping DEPs whose expression was inhibited by SMZCL and rapamycin. KEGG enrichment analysis showed that necroptosis process was a key pathway for SMZCL to treat NA airway inflammation. IHC and WB results confirmed that SMZCL and rapamycin inhibited the phosphorylation of RIPK1, PIPK3 and MLKL. In vitro, ATG5 and LC3 proteins were obviously increased while p-mTOR expression was inhibited after amygdalin treatment. ConclusionSMZCL attenuated airway inflammation in NA mainly through inhibition of the mTOR pathway, along with inhibition of the necroptosis pathway regulated by the RIPK1/RIPK3/MLKL axis and inhibition of Mzb1 and Rab3ip expression.

Usage of Foley’s catheter in pediatric lung isolation, whole lung lavage: A case report

This case report delves into pediatric lung isolation challenges and innovations in managing patients with pulmonary alveolar proteinosis undergoing whole lung lavage. The central focus is on a 5-year-old girl who initially encountered intraoperative complications, including bilateral pneumothorax and pulmonary edema. However, a subsequent attempt employing a Foley’s catheter for lung isolation proved successful, with the patient displaying marked postoperative improvements. The case offers valuable insights into the intricate balance of anesthesia, ventilatory parameters, and the novel use of common medical equipment, like the Foley’s catheter, for specialized procedures in pediatric pulmonology.