Bronchoalveolar Fluid Research Articles

Piperine attenuates production of inflammatory biomarkers, oxidative stress and neutrophils in lungs of cigarette smoke-exposed experimental mice

Cigarette smoking (CS) induced chronic obstructive pulmonary disease (COPD) is third leading cause of global pulmonary health issue. Currently, there is no effective therapeutic strategy to control perpetuating inflammatory response in COPD. The aim of current study was to investigate the protective effects of piperine in CS-induced murine model of COPD. Exposure of experimental animals to cigarettes smoke twice daily for 10 weeks resulted in increased production of pro-inflammatory mediators including IL-1β, IL-8, TNF-α, LTB-4 and neutrophil elastase (NE) in bronchoalveolar (BAL) fluid. Oxidative stress biomarkers, malondialdehyde (MDA), myeloperoxidase (MPO), nitric oxide and MMP9 also significantly increased in CS-control mice lungs as compared to normal control group (P < 0.001). Moreover, CS also increased recruitment of inflammatory cells, neutrophils and macrophages into BAL fluid and altered breathing rate and tidal volume as compared to normal control mice. However, oral treatment of CS-exposed animals with test drug, piperine (10 mg/kg, b.w. or 20 mg/kg, b.w.) or reference standard methyl prednisolone (3.25 mg/kg, b.w.) for 21 days or 7 days, respectively, significantly (p < 0.05, 0.01, 0.001) improved pulmonary function and reversed CS-induced production of cytokines, oxidative stress biomarkers and other biochemicals as compared to CS-exposed control mice. Further, In silico molecular docking studies exhibited attractive binding affinity of piperine for human neutrophil elastase, MMP-9, MPO and IL-8 receptors. Findings of our study suggest protective effect of piperine in experimental animals by repressing CS-induced infiltration of inflammatory cells and thereby exaggerated production of pro-inflammatory mediators and oxidative stress.

Analytics Pipeline for Visualization of Single Cell RNA Sequencing Data from Brochoaveolar Fluid in COVID-19 Patients: Assessment of Neuro Fuzzy-C-Means and HDBSCAN.

Since the mutation in SARS-COV2 poses new challenges in designing vaccines, it is imperative to develop advanced tools for visualizing the genetic information. Specially, it remains challenging to address the patient-to-patient variability and identify the signature for severe/critical conditions. In this endeavor we analyze the large-scale RNA-sequencing data collected from broncho-alveolar fluid. In this work, we have used PCA and tSNE for the dimension-reduction. The novelty of the current work is to depict a detailed comparison of k-means, HDBSAN and neuro-fuzzy method in visualization of high-dimension data on gene expression. Clinical Relevance- The subpopulation profiling can be used to study the patient-to patient variability when infected by SARS-COV-2 and its variants. The distribution of cell types can be relevant in designing new drugs that are targeted to control the distribution of epithelial cells T cells and macrophages.

Diagnostic Accuracy of Bronchoalveolar Lavage in Diagnosis of Ple-ural Tuberculosis among Suspected Cases of Pleural TB

Aim: To determine the accuracy of bronchoalveolar lavage in diagnosis of pleural tuberculosis among suspected cases of pleural tuberculosis using biopsy. Study design: Descriptive, cross-sectional. Place and duration of study: Department of Chest Medicine, JPMC, Karachi from 1st August 2017 to 31st January 2018. Methodology: One hundred and thirty six patients with cases of pleural tuberculosis of age 18-55 years and both genders were included. Patients with anti-tuberculosis drugs, pneumonia, tonsillitis, pharyngitis, obstructive pulmonary disease and platelet function disorders, thrombocytopenia or coagulation factors disorder were excluded. 30-50 ml of bronchoalveolar fluid was carried out through fiberoptic bronchoscopy. Three milliliters of 4% lignocaine was used as pre-medication and 3 ml of 2% lignocaine was used during bronchoscopy. Results: In BAL positive patients, 73 (true positive) had pleural tuberculosis and 9 (false positive) had no pleural tuberculosis on biopsy. Among 54, BAL negative patients, 5 (false negative) had pleural tuberculosis on biopsy whereas 49 (true negative) had no pleural tuberculosis on biopsy. Overall sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of BAL in diagnosis of pleural tuberculosis among suspected cases of pleural tuberculosis using biopsy as gold standard was 93.59%, 84.48%, 89.02%, 90.74% and 89.71% respectively. Conclusion: The diagnostic accuracy of bronchoalveolar lavage in diagnosis of pleural tuberculosis among suspected cases of pleural tuberculosis is quite high. Keywords: Pleural tuberculosis, Bronchoalveolar lavage, Sensitivity

PSEUDOTUMORAL PULMONARY TB MASS: WHAT IS THE PLACE OF SURGERY FOR DIAGNOSIS?

TYPE: Abstract TOPIC: Cardiothoracic Surgery PURPOSE: The pseudotumoral form of pulmonary tuberculosis (PTPT) is extremely rare. It constitutes a diagnosis dilemma by simulating pulmonary cancer. Surgical investigation is required when non-invasive modalities of diagnostic don’t identify the PTPT. METHODS: Sixteen immunocompetent patients presenting a PTPT, were operated in the Thoracic and Cardiovascular Surgery Department of Ariana in Tunisia between 1997 and 2020. RESULTS: They were 14 men and 2 women. The average age was 53 years. Two patients had a history of treated pulmonary tuberculosis. Eleven patients were smokers. The median overall diagnostic delay was 6 months. Productive cough (n=4) and chest pain (n=4) were the major symptoms. The chest X-ray revealed a dishomogeneous opacity with an irregular outline in 75% of cases. The computed tomography of chest showed a tissue mass mimickig malignancy in 10 cases and a suspicious parenchymal condensation in 2 cases. The most common site involved is the upper right lobe (10 cases). Cavitation was seen in 3 cases. The bronchoscopy showed a stenosing bronchial wall infiltration in 5 cases. All bronchial biopsies were inconclusive. Sputum and bronchoalveolar fluid acid fast bacilli smear and culture were negative. Through a posterolateral thoracotomy, we performed a lobectomy (n=12), wedge resection (n=2) and tumor biopsy (n=2). Histological exam concluded to pulmonary tuberculosis. All patients received anti-bacillary therapy with good clinico-radiological evolution. CONCLUSIONS: The positive diagnosis of PTPT is difficult. Surgery remains the reliable means of diagnosis for initiating early medical management. CLINICAL IMPLICATIONS: Diagnosis of PTPT can be delayed and lead to surgical resection DISCLOSURE: Nothing to declare. KEYWORD: pseudotumoral, lung, tuberculosis, surgery

Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine.

Despite success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability remain. Molecular adjuvants targeting pattern recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulates various PRRs, including toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors. We hypothesized that targeting PRRs using molecular adjuvants on nanoparticles (NPs) along with a stabilized spike protein antigen could stimulate broad and efficient immune responses. Adjuvants targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer NPs were combined with the S1 subunit of spike protein and assessed in vitro with isogeneic mixed lymphocyte reactions (isoMLRs). For in vivo studies, the adjuvant-NPs were combined with stabilized spike protein or spike-conjugated NPs and assessed using a two-dose intranasal or intramuscular vaccination model in mice. Combination adjuvant-NPs simultaneously targeting TLR and RIG-I receptors (MPLA+PUUC, CpG+PUUC, and R848+PUUC) differentially induced T cell proliferation and increased proinflammatory cytokine secretion by APCs in vitro. When delivered intranasally, MPLA+PUUC NPs enhanced CD4+CD44+ activated memory T cell responses against spike protein in the lungs while MPLA NPs increased anti-spike IgA in the bronchoalveolar (BAL) fluid and IgG in the blood. Following intramuscular delivery, PUUC NPs induced strong humoral immune responses, characterized by increases in anti-spike IgG in the blood and germinal center B cell populations (GL7+ and BCL6+ B cells) in the draining lymph nodes (dLNs). MPLA+PUUC NPs further boosted spike protein-neutralizing antibody titers and T follicular helper cell populations in the dLNs. These results suggest that protein subunit vaccines with particle-delivered molecular adjuvants targeting TLR4 and RIG-I could lead to robust and unique route-specific adaptive immune responses against SARS-CoV-2.

Abstract A026: Altered immune landscape in aging lungs contributes to malignant evolution

Abstract Aging is a major risk factor in increased lung cancer incidence. While most research has focused on age-associated mutation accumulation to explain the late-life increase in cancer incidence, there are tissue environmental forces that both impede and promote cancer evolution. Just as organismal evolution is known to be driven by environmental changes, cellular (somatic) evolution in our bodies is similarly driven by changes in tissue environments. Environmental change promotes selection for new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven alterations in tissues drives selection for adaptive mutations, and some of these mutations can confer malignant phenotypes. Chronic, low-level inflammation has been associated with aging, termed inflammaging, yet how age-associated changes in lung tissue microenvironments contribute to increased lung cancer incidence has remained largely unknown. Since chronic inflammation has been shown to contribute to tumor development, we hypothesized that inflammaging contributes to increased oncogenic adaptation in the lung. Using either viral delivery of CRISPR constructs to mediate EML4-ALK translocations or ectopic expression of KRAS-G12D, we showed increased adenoma formation in old mice. Importantly, in the EML4-ALK model, we showed that the overexpression of alpha-1 antitrypsin (AAT) in old mice resulted in lower adenoma counts compared to their old wild type counterparts. Flow cytometric analysis of immune cells isolated from bronchoalveolar fluid of young and old mice showed an altered immune landscape, such as increased neutrophils, gamma delta T cells, and Foxp3+ regulatory T cells. Furthermore, analysis of the single-cell RNAseq data from Tabula Muris Consortium demonstrated increased exhaustion markers in the CD8+ T cells and regulatory T cells. Separately, Gene Set Enrichment Analysis (GSEA) of the differential gene expressions of lung epithelial cells isolated from young and old mice revealed enriched pathways related to immune activation and inflammatory response, and immune-suppression markers. Lastly, bulk RNA-seq from lungs of young, old, and old mice overexpressing AAT revealed increased immune cell exhaustion markers and that the overexpression of AAT partially reversed this increase. Finally, analysis of Genotype-Tissue Expression (GTEx) data comparing gene expressions in lungs of young and old humans similarly showed enriched pathways related to immune activation and increased T cell exhaustion markers in the elderly. In addition, using deconvolution methods CiberSort and xCell, we demonstrated altered innate and adaptive immune cell populations, for example, increased neutrophils and regulatory T cells, that are associated with advanced age, similar to aging mice. In conclusion, we showed that there is an exhausted immune microenvironment in aging lungs, that inflammation contributes to the increased tumor initiation, and that decreasing inflammation could decrease the lung tumor incidence by reactivating the immune system. Citation Format: Shi Biao Chia, Catherine Pham-Danis, Hannah Scarborough, Nathaniel Little, Etienne P. Danis, Andrew E. Goodspeed, Charles Dinarello, James DeGregori. Altered immune landscape in aging lungs contributes to malignant evolution [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A026.

Challenges in Metabolomics-Based Tests, Biomarkers Revealed by Metabolomic Analysis, and the Promise of the Application of Metabolomics in Precision Medicine.

Metabolomics helps identify metabolites to characterize/refine perturbations of biological pathways in living organisms. Pre-analytical, analytical, and post-analytical limitations that have hampered a wide implementation of metabolomics have been addressed. Several potential biomarkers originating from current targeted metabolomics-based approaches have been discovered. Precision medicine argues for algorithms to classify individuals based on susceptibility to disease, and/or by response to specific treatments. It also argues for a prevention-based health system. Because of its ability to explore gene–environment interactions, metabolomics is expected to be critical to personalize diagnosis and treatment. Stringent guidelines have been applied from the very beginning to design studies to acquire the information currently employed in precision medicine and precision prevention approaches. Large, prospective, expensive and time-consuming studies are now mandatory to validate old, and discover new, metabolomics-based biomarkers with high chances of translation into precision medicine. Metabolites from studies on saliva, sweat, breath, semen, feces, amniotic, cerebrospinal, and broncho-alveolar fluid are predicted to be needed to refine information from plasma and serum metabolome. In addition, a multi-omics data analysis system is predicted to be needed for omics-based precision medicine approaches. Omics-based approaches for the progress of precision medicine and prevention are expected to raise ethical issues.

Cortactin loss protects against hemin-induced acute lung injury in sickle cell disease.

In patients with sickle cell disease (SCD), acute chest syndrome (ACS) is a common form of acute lung injury and a major cause of morbidity and mortality. The pathophysiology of ACS is complex, and hemin, the prosthetic moiety of hemoglobin, has been implicated in endothelial cell (EC) activation and subsequent acute lung injury (ALI) and ACS in vitro and in animal studies. Here, we examined the role of cortactin (CTTN), a cytoskeletal protein that regulates EC function, in response to hemin-induced ALI and ACS. Cortactin heterozygous (Cttn+/-) mice (n = 8) and their wild-type siblings (n = 8) were irradiated and subsequently received bone marrow cells (BMCs) extruded from the femurs of SCD mice (SS) to generate SS Cttn+/- and SS CttnWT chimeras. Following hemoglobin electrophoretic proof of BMC transplantation, the mice received 35 µmol/kg of hemin. Within 24 h, surviving mice were euthanized, and bronchoalveolar fluid (BAL) and lung samples were analyzed. For in vitro studies, human lung microvascular endothelial cells (HLMVECs) were used to determine hemin-induced changes in gene expression and reactive oxygen species (ROS) generation in cortactin deficiency and control conditions. When compared with wild-type littermates, the mortality for SS Cttn+/- mice trended to be lower after hemin infusion and these mice exhibited less severe lung injury and less necroptotic cell death. In vitro studies confirmed that cortactin deficiency is protective against hemin-induced injury in HMLVECs, by decreasing protein expression of p38/HSP27, improving cell barrier function, and decreasing the production of ROS. Further studies examining the role of CTTN in ACS are warranted and may open a new avenue of potential treatment for this devastating disease.

Virus-induced and bacteria-induced exacerbations of chronic obstructive pulmonary disease caused by industrial aerosols or tobacco smoke exposure

Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are associated with disease progression and increased risk of death. We need to better understand the phenotypes of AECOPD to improve treatment strategies. The main triggers of COPD exacerbations are viral and bacterial infections.The aim is to characterize the viral, bacterial, and viral-bacterial phenotypes of acute exacerbations in patients with COPD caused by industrial aerosol exposure or tobacco smoke.Methods. 180 subjects with established moderate and severe COPD who met the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria, 2020 – 2021, and were hospitalized with AECOPD, were enrolled in this prospective observational study. The virus-induced, bacteria-induced, and virus-bacteria-induced AECOPD strata (n = 60 each) were formed. Each stratum included 30 patients with occupational COPD and 30 patients with COPD caused by tobacco smoke. Virus-induced AECOPDs were diagnosed by PCR of bronchoalveolar fluid. Length of hospital stay, symptoms, lung function, mean pulmonary artery pressure (mPAP), and type of inflammation were assessed. Cox proportional hazard regression was used to examine the relationships.Results. The length of hospital stay was highest in patients with virus-induced and virus-bacteria-induced exacerbations of occupational COPD, being equal to (Me, IQR) 16.5 (14 – 18) and 18 (16 – 20) days. The virus-induced exacerbations in occupational COPD and in COPD caused by tobacco smoke featured the highest bronchodilation coefficient, 10.9 (9.8 – 11,5)% and 9.2 (8.3 – 10.3)%, respectively, decrease in the diffusing capacity of the lungs (DLCO/Va) by 42 (40 – 45)% and 49 (47 – 52)%, increase in mPAP by 44 (39 – 45) и 33 (29 – 38) mmHg, and eosinophilic inflammation with blood eosinophil count of 425 (385 –527) and 350 (310 – 391) cells per μl (р &gt; 0.01). Virus-bacteria-induced AECOPD in occupational COPD and in COPD caused by smoke were characterized by decrease in FEV1 by 40.2 (36.6 – 42.2)% and 31.0 (28.1 – 33.6%), decrease in DLCO/Va by 48 (44 – 50)% and 37 (35 – 41)%, increase in mPA by 43 (38 – 46) and 50 (45 – 54) mmHg, and eosinophilic-neutrophilic inflammation in 63.3 and 66.6% of patients. The mid-range FEV1, highest DLCO/Va, and neutrophilic inflammation were seen in patients with bacteria-induced AECOPD.Conclusion. Exacerbations of occupational COPD are characterized by more severe functional impairment and inflammation with high eosinophil count when these exacerbations have viral origin.

Segmental Bronchial Allergen Challenge Elicits Distinct Metabolic Phenotypes in Allergic Asthma

Asthma is a complex syndrome associated with episodic decompensations provoked by aeroallergen exposures. The underlying pathophysiological states driving exacerbations are latent in the resting state and do not adequately inform biomarker-driven therapy. A better understanding of the pathophysiological pathways driving allergic exacerbations is needed. We hypothesized that disease-associated pathways could be identified in humans by unbiased metabolomics of bronchoalveolar fluid (BALF) during the peak inflammatory response provoked by a bronchial allergen challenge. We analyzed BALF metabolites in samples from 12 volunteers who underwent segmental bronchial antigen provocation (SBP-Ag). Metabolites were quantified using liquid chromatography-tandem mass spectrometry (LC–MS/MS) followed by pathway analysis and correlation with airway inflammation. SBP-Ag induced statistically significant changes in 549 features that mapped to 72 uniquely identified metabolites. From these features, two distinct inducible metabolic phenotypes were identified by the principal component analysis, partitioning around medoids (PAM) and k-means clustering. Ten index metabolites were identified that informed the presence of asthma-relevant pathways, including unsaturated fatty acid production/metabolism, mitochondrial beta oxidation of unsaturated fatty acid, and bile acid metabolism. Pathways were validated using proteomics in eosinophils. A segmental bronchial allergen challenge induces distinct metabolic responses in humans, providing insight into pathogenic and protective endotypes in allergic asthma.

Lipopolysaccharide from the Cyanobacterium Geitlerinema sp. Induces Neutrophil Infiltration and Lung Inflammation.

Glucocorticoid-resistant asthma, which predominates with neutrophils instead of eosinophils, is an increasing health concern. One potential source for the induction of neutrophil-predominant asthma is aerosolized lipopolysaccharide (LPS). Cyanobacteria have recently caused significant tidal blooms, and aerosolized cyanobacterial LPS has been detected near the cyanobacterial overgrowth. We hypothesized that cyanobacterial LPS contributes to lung inflammation by increasing factors that promote lung inflammation and neutrophil recruitment. To test this hypothesis, c57Bl/6 mice were exposed intranasally to LPS from the cyanobacterium member, Geitlerinema sp., in vivo to assess neutrophil infiltration and the production of pro-inflammatory cytokines and chemokines from the bronchoalveolar fluid by ELISA. Additionally, we exposed the airway epithelial cell line, A549, to Geitlerinema sp. LPS in vitro to confirm that airway epithelial cells were stimulated by this LPS to increase cytokine production and the expression of the adhesion molecule, ICAM-1. Our data demonstrate that Geitlerinema sp. LPS induces lung neutrophil infiltration, the production of pro-inflammatory cytokines such as Interleukin (IL)-6, Tumor necrosis factor-alpha, and Interferongamma as well as the chemokines IL-8 and RANTES. Additionally, we demonstrate that Geitlerinema sp. LPS directly activates airway epithelial cells to produce pro-inflammatory cytokines and the adhesion molecule, Intercellular Adhesion Molecule-1 (ICAM-1), in vitro using the airway epithelial cell line, A549. Based on our findings that use Geitlerinema sp. LPS as a model system, the data indicate that cyanobacteria LPS may contribute to the development of glucocorticoid-resistant asthma seen near water sources that contain high levels of cyanobacteria.

Sandwich-type homogeneous chemiluminescence immunoassay based on nanoparticle toward detection of Aspergillus galactomannan antigen

Aspergillus species continue to be an important cause of life-threatening infection in immunocompromised patients and galactomannan (GM) is a popular biomarker in the diagnosis of invasive aspergillosis (IA). Here we developed and validated an amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) for serum and bronchoalveolar fluid (BALF) GM based on this approach. Technological processes and reaction conditions were optimized. Study assessments included reproducibility, accuracy, stability, and cross reactivity experiments. Method comparisons with the commercial Platelia Aspergillus enzyme immunoassay (Bio-Rad Laboratories) were performed using 201 clinical serum and BALF samples. Under the optimized conditions, the total runtime of the AlphaLISA method was 40 min with simple operation. The percent coefficient variations (CVs%) were lower than 15%, and the recoveries were in the range of 90–110%. Of note, the proposed assay exhibited acceptable stability and did not display cross-reactivity with non-Aspergillus pathogens. Compared with the results from the Platelia kit, there was a satisfied overall qualitative agreement of 97.5% and overall quantitative correlation coefficient of 0.59. In all, we have successfully developed an alternative novel homogeneous nanoparticle-based immunoassay, which has shorter incubation time and easier protocol than the ones of conventional ELISA. It could serve as an alternative to the well-established Platelia assay for measurement of GM in serum and BALF.

Incorporating Biomarkers in COPD Management: The Research Keeps Going.

Globally, chronic obstructive pulmonary disease (COPD) remains a major cause of morbidity and mortality, having a significant socioeconomic effect. Several molecular mechanisms have been related to COPD including chronic inflammation, telomere shortening, and epigenetic modifications. Nowadays, there is an increasing need for novel therapeutic approaches for the management of COPD. These treatment strategies should be based on finding the source of acute exacerbation of COPD episodes and estimating the patient’s own risk. The use of biomarkers and the measurement of their levels in conjunction with COPD exacerbation risk and disease prognosis is considered an encouraging approach. Many types of COPD biomarkers have been identified which include blood protein biomarkers, cellular biomarkers, and protease enzymes. They have been isolated from different sources including peripheral blood, sputum, bronchoalveolar fluid, exhaled air, and genetic material. However, there is still not an exclusive biomarker that is used for the evaluation of COPD but rather a combination of them, and this is attributed to disease complexity. In this review, we summarize the clinical significance of COPD-related biomarkers, their association with disease outcomes, and COPD patients’ management. Finally, we depict the various samples that are used for identifying and measuring these biomarkers.

Correlation between Increased Monocyte Chemotactic Protein 1 Level and Lung Function in Patients with Asthma

Background: The altered expression of monocyte chemotactic protein 1 (MCP1) in bronchoalveolar fluid was observed in patients and animal models of allergic asthma. However, the correlation between induced sputum MCP1 level and asthma clinical features remains poorly understood. Objective: This study aims to explore the relationship of MCP1 protein expression in induced sputum with Th2 inflammation and asthma clinical features. Methods: MCP1 protein expression in induced sputum and serum was detected by ELISA in patients with asthma, and its correlation with Th2 inflammation and lung function was analyzed. The effect of inhaled corticosteroids (ICSs) on MCP1 expression was also evaluated. Results: The MCP1 level in induced sputum (p = 0.0006) and serum (p = 0.0035) was markedly increased and negatively correlated with forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC)% (r = −0.3674, p = 0.0001) and PC-20 (r = −0.5746, p < 0.0001) in patients with asthma. The area under the curve (AUC) of MCP1 level receiver operating characteristic curve in induced sputum and serum was 0.7134 (p = 0.0007) and 0.7589 (p = 0.0042), respectively. The patients with asthma were grouped into four according to their induced sputum MCP1 level and Th2 signature categories (Th2^Hi MCP1^Hi, Th2^Hi MCP1^Low, Th2^Low MCP1^Hi, and Th2^Low MCP1^Low). The Th2^Low MCP1^Hi subgroup had the lowest FEV1/FVC% and histamine concentration required to cause a 20% decline in FEV1. After 4 weeks of ICS treatment, the MCP1 levels in induced sputum and serum were significantly reduced. Conclusions: The MCP1 level in induced sputum and serum increased in patients with asthma but decreased after ICS treatment. The Th2^Low MCP1^Hi subgroup had the worst lung function and highest airway hyperresponsiveness. The combination of MCP1 level in induced sputum and Th2 inflammation defines a new phenotype that can be used to predict lung function and treatment response in patients with asthma.

House Dust Mite Aeroallergen Suppresses Leukocyte Phagocytosis and Netosis Initiated by Pneumococcal Lung Infection.

Asthmatics are highly susceptible to developing lower respiratory tract infections caused by Streptococcus pneumoniae (SPN, the pneumococcus). It has recently emerged that underlying allergic airway disease creates a lung microenvironment that is defective in controlling pneumococcal lung infections. In the present study, we examined how house dust mite (HDM) aeroallergen exposure altered immunity to acute pneumococcal lung infection. Alveolar macrophage (AM) isolated from HDM-exposed mice expressed alternatively activated macrophage (AAM) markers including YM1, FIZZ1, IL-10, and ARG-1. In vivo, prior HDM exposure resulted in accumulation of AAMs in the lungs and 2-log higher bacterial titres in the bronchoalveolar (BAL) fluid of SPN-infected mice (Day 2). Acute pneumococcal infection further increased the expression of IL-10 and ARG1 in the lungs of HDM-exposed mice. Moreover, prior HDM exposure attenuated neutrophil extracellular traps (NETs) formation in the lungs and dsDNA levels in the BAL fluid of SPN-infected mice. In addition, HDM-SPN infected animals had significantly increased BAL fluid cellularity driven by an influx of macrophages/monocytes, neutrophils, and eosinophils. Increased lung inflammation and mucus production was also evident in HDM-sensitised mice following acute pneumococcal infection, which was associated with exacerbated airway hyperresponsiveness. Of note, PCV13 vaccination modestly reduced pneumococcal titres in the BAL fluid of HDM-exposed animals and did not prevent BAL inflammation. Our findings provide new insights on the relationship between pneumococcal lung infections and allergic airways disease, where defective AM phagocytosis and NETosis are implicated in increased susceptibility to pneumococcal infection.

Reactivation of Herpes Simplex Virus Type 1 (HSV-1) Detected on Bronchoalveolar Lavage Fluid (BALF) Samples in Critically Ill COVID-19 Patients Undergoing Invasive Mechanical Ventilation: Preliminary Results from Two Italian Centers.

Reactivation of herpes simplex virus type 1 (HSV-1) has been described in critically ill patients with coronavirus disease 2019 (COVID-19) pneumonia. In the present two-center retrospective experience, we primarily aimed to assess the cumulative risk of HSV-1 reactivation detected on bronchoalveolar fluid (BALF) samples in invasively ventilated COVID-19 patients with worsening respiratory function. The secondary objectives were the identification of predictors for HSV-1 reactivation and the assessment of its possible prognostic impact. Overall, 41 patients met the study inclusion criteria, and 12/41 patients developed HSV-1 reactivation (29%). No independent predictors of HSV-1 reactivation were identified in the present study. No association was found between HSV-1 reactivation and mortality. Eleven out of 12 patients with HSV-1 reactivation received antiviral therapy with intravenous acyclovir. In conclusion, HSV-1 reactivation is frequently detected in intubated patients with COVID-19. An antiviral treatment in COVID-19 patients with HSV-1 reactivation and worsening respiratory function might be considered.

РОЛЬ ИММУНОЛОГИЧЕСКИХ ФАКТОРОВ В НЕБЛАГОПРИЯТНОМ ИСХОДЕ ХРОНИЧЕСКОЙ ОБСТРУКТИВНОЙ БОЛЕЗНИ ЛЕГКИХ

Aim. The aim of the research was to study the role of immunological mechanisms in the unfavorable outcome of chronic obstructive pulmonary disease (COPD). Material and methods. The research included 116 patients hospitalized in 2005-2006 in the pulmonology department of patients with the exacerbation of mild and moderate COPD. Research protocol: general clinical and special (immunological) methods of examination were performed in patients with COPD. Instrumental research methods included: spirography, ECG, EchoCG, fibrobronchoscopy, chest X-ray. After diagnostic bronchoscopy, bronchoalveolar fluid (BAF) was taken for cytological and immunological examination. On the first and second days of inpatient treatment, patients underwent immunological studies: immunophenotyping of mononuclear cells (MNC), assessment of phagocytic activity of leukocytes (latex test, nitroblue tetrazolium (NBT) test) in blood and BAF, determination of the content of immunoglobulins in blood serum and BAF, study of cytokine levels in blood serum, BAF and MNC culture supernatant under conditions of their spontaneous production and activation in the mitogen-stimulated (phytohemagglutinin "Difco" 5 mg/ml) proliferation. Statistical data processing was carried out using the Statistica 10.0 program. In 2020, the long-term survival of COPD patients was assessed. The cohort of patients was divided into two groups. Survivors were included in the first group (n=44), and patients who died by 2020 were included in the other group (n=72). The retrospective comparison of the studied indicators was determined at the time of COPD exacerbation in 2005- 2006 in these patient groups. Results and discussion. In the surviving patients, the BAF cytogram was distinguished by the higher macrophage content and the increased number of NBT-positive cells in this population with the reduced neutrophil content in both blood and BAF. The deceased had elevated levels of IgM and IgA in the blood serum and BAF. The results of the study of cytokine production in MNC cultures in vitro indicate that the ratio of IFN-γ/IL-4 in the conditions of spontaneous cytokine production was higher in the deceased compared with the indicators of the surviving patients. This fact indicates the polarization of the immune response of the deceased in the direction of the cell type mediated by Th1 cells, which is confirmed by the peculiarities of the BAF cytokine profile in the deceased – the significant increase in the value of IFN-γ/IL-4 relative to the indicators in the survivors. Conclusion. The unfavorable outcome of COPD is associated with the increase in the number of neutrophil cells in BAF, in the blood, with the predominance of Th1-type activation of adaptive immunity at the local level and activation of both cellular and humoral mechanisms of adaptive immunity at the systemic level. The increased activity of innate immunity in COPD exacerbation, manifested by the increase in the number and metabolic activity of macrophages, is associated with the long-term survival of patients.

Lung microbiota features of stage III and IV non-small cell lung cancer patients without lung infection.

BackgroundThe microbial community affects the occurrence, development, metastasis and treatment response of cancers. But the detailed role and characteristics of lung microbiota (LM) in non-small cell lung cancer (NSCLC) are not fully known. For NSCLC associated microbiota analysis, it is valuable to combine multiple levels of detection, e.g., tumor, blood plasma, and bronchoalveolar fluid (BALF), but not single tissues.MethodsThis study collected above three sample types from NSCLC patients free from lung infection and aimed to describe their LM features using sequencing techniques. All patients diagnosed at the Department of Oncology in Shijiazhuang People’s Hospital with stage III or IV NSCLC from May 2019 to April 2020 were enrolled. All 37 pieces of tumor tissues and 6 blood samples were sent for pathogen targeted sequencing; for the BALF samples, 4 were used for pathogen targeted sequencing and 2 were sent for 16S ribosomal DNA (rDNA) sequencing.ResultsWe detected 49 pathogenic microorganisms (PMs) in the 37 tumor samples, 28 PMs in the 4 BALF samples, and 14 PMs in the 6 plasma samples. Overall, there were 5 common PMs in 3 types of samples. Between the tumor and BALF samples, there were another 11 common elements. In the 5 tumor-plasma pairs, the presence of a specific PM in blood was not necessarily consistent with that in the tumor. In the tumor-BALF pairs, the PM diversity was dramatically higher in the BALF than in the tumor. The PMs detected in the BALF could largely cover the PMs in the tumor. In the BALF 16S rDNA sequencing, there were 82 common operational taxonomic units (OTUs), and the microbiota in the BALF of advanced NSCLC patients exhibited some similarity.ConclusionsThis study showed the unique features of LM. The amount of intra-tumoral PMs was not necessarily consistent with that in the blood, but there was an obvious correlation between the intra-tumoral microbiota and that in the BALF. It is convenient and non-invasive to obtain BALF. Detection of LM classification and abundance in the BALF may help evaluate the severity of NSCLC.

ТЕХНОЛОГИИ GX ДЛЯ БЫСТРОЙ ДИАГНОСТИКИ ТУБЕРКУЛЕЗА В УЧРЕЖДЕНИЯХ ОБЩЕЙ ЛЕЧЕБНОЙ СЕТИ

Introduction. There is still a high percentage of tuberculosis detection by the number of complaints to the general medical network. Differential diagnosis is often performed in pulmonology departments using a standard diagnostic minimum. Reducing the time of diagnosis of tuberculosis is possible with the introduction of molecular genetic methods in the standards of primary examination. Aim. The aim is to determine the possibility of improving the detection of pulmonary tuberculosis using molecular genetic methods by the example of GeneXpert MTB/RIF cartridge technology in the pulmonology department of the general treatment network of healthcare. Material and methods. GeneXpert MTB/ RIF cartridge technology (MGM) was used in 159 patients with high risks of tuberculosis, characteristic changes on the X-ray image in addition to the standard study for the search for M. tuberculosis in biological material (sputum or bronchoalveolar fluid). Аccording to verified diagnoses, two groups were formed: 1st - tuberculosis (n = 119), second - pneumonia (n = 40). Statistical processing was carried out using the IBM SPSS Statistics 22.0 statistical software package. Results and discussions. In the differential diagnosis of tuberculosis in patients of the pulmonology department, there is a commonality of criteria for age (p=0.066), gender (p=0.249), unemployment (p=0.452), HIV infection (p=0.115), drug addiction (p=0.066), hepatitis (p=0.076). Positive results were on smear microscopy with sputum staining by Ziehel- Neelsen 18.9%, bronchoalveolar fluid 31%. The method of GeneXpert MTB/RIF cartridge technology showed a high sensitivity of 100% and specificity of 99% in all cases of a verified diagnosis of «tuberculosis» using culture methods. Conclusion. In patients with pneumonia who have risk factors for tuberculosis, when the GeneXpert MTB/RIF cartridge technology for M. tuberculosis search is included in the standard diagnostic minimum in the general medical network, the effectiveness of tuberculosis detection increases while reducing the time of diagnosis

Clinical and bacteriological analysis of respiratory tract infections in sheltered dogs and determination of antibacterial treatment options

After canine infectious respiratory disease complex-CIRDC, only bacterial pneumonia or accompanied with CIRDC has a higher proportion in respiratory disease in stray dogs. Management of these respiratory problems in terms of both treatment and prevention in shelters has a big importance for animal welfare. With this purpose, the present study evaluates bacterial pneumonia in terms of clinical, bacteriological and the antibacterial treatment options in 100 sheltered dogs with respiratory tract infection symptoms. In all dogs, status of respiratory disease and treatment efficacy were evaluated by haematological analysis and clinical scores. Haematological analyses showed that all of the dogs suffered leucocytosis before treatment. Health status of all animals before, during and after treatment were evaluated according to nine clinical scores include clinical condition, body temperature, respiratory and heart rates per minutes, nasal discharge, tracheal sensitivity, mucous membranes, coughing, auscultation. For bacteriological analysisand antimicrobial susceptibility tests, Bronchoalveolar lavage-BAL fluids were obtained from all dogs, twice. The bacterial agents isolated in the present study were Bordetella spp. (38.98%), Mycoplasma spp. (21.19%), Klebsiella spp. (16.10%), E. coli (5.93%), S. aureus (5.08%) and Pasteurella spp. (4.24%). Susceptibility tests were performed by using the disc diffusion method for Enrofloxacin (ENR), Trimethoprim/Sulpha (TS), Chloramphenicol (C), Amoxicillin clavulanate (AC), and Erythromycin (E) in all cases. Bordetella spp. isolated from 46 cases were found to be most susceptible to ENR (21/46 = 46%), TS (12/46 = 26%), and C (11/46 = 24%). Mycoplasma spp. were isolated from 25 cases and were found to be susceptible to C (14/25 = 56%), TS (8/25 = 32%), and ENR (3/25 = 12%). Klebsiella spp. were isolated from 19 cases and the antibiotics most effective were ascertained as C (9/19 = 47%), ENR (9/19 = 47%), and TS (1/19 = 5%).The results showed that clinical scores could be useful in the diagnosis and monitoring of respiratory tract diseases in sheltered dogs. Besides, in the light of the findings of presented study, enrofloxacin, chloramphenicol, and trimethoprim/sulpha were proven efficient against to bacterial isolates in sheltered dogs in the treatment of bacterial pneumonia. Antibacterial therapy should be conducted by antibiotic sensitivity test. But, in the cases this is not possible, antibiotic choice may contain enrofloxacin, trimethoprim/sulpha. If it is not forbidden to use for companion animals by administrations, chloramphenicol may also be thought as alternative.