Blood And Bronchoalveolar Lavage Research Articles

Randomised, double-blind, controlled phase 1 trial of the candidate tuberculosis vaccine ChAdOx1-85A delivered by aerosol versus intramuscular route

A BCG booster vaccination administered via the respiratory mucosa may establish protective immune responses at the primary site of Mycobacterium tuberculosis infection. The primary objective of this trial was to compare the safety and immunogenicity of inhaled versus intramuscular administered ChAdOx1-85A. We conducted a single-centre, randomised, double-blind, controlled phase 1 study (Swiss National Clinical Trials Portal number SNCTP000002920). After a dose-escalation vaccination in nine BCG-vaccinated healthy adults, a dose of 1×1010 vp of ChAdOx1-85A was administered to twenty BCG-vaccinated adults that were randomly allocated (1:1) into two groups: aerosol ChAdOx1-85A with intramuscular saline placebo or intramuscular ChAdOx1-85A with aerosol saline placebo, using block randomisation. A control group of ten BCG-naïve adults received aerosol ChAdOx1-85A at the same dose. Primary outcomes were solicited and unsolicited adverse events (AEs) up to day 16 post-vaccination and Serious AEs (SAEs) up to 24 weeks; secondary outcomes were cell-mediated and humoral immune responses in blood and bronchoalveolar lavage (BAL) samples. Both vaccination routes were well tolerated with no SAEs. Intramuscular ChAdOx1-85A was associated with more local AEs (mostly pain at the injection site) than aerosol ChAdOx1-85A. Systemic AEs occurred in all groups, mainly fatigue and headaches, without differences between groups. Respiratory AEs were not different between BCG-vaccinated groups. Aerosol ChAdOx1-85A vaccination induced Ag85A BAL and systemic cellular immune responses with compartmentalisation of the immune responses: aerosol ChAdOx1-85A induced stronger BAL cellular responses, particularly IFNγ/IL17+CD4+T cells; intramuscular ChAdOx1-85A induced stronger systemic cellular and humoral responses. Inhaled ChAdOx1-85A was well-tolerated and induced lung mucosal and systemic Ag85A-specific T-cell responses. These data support further evaluation of aerosol ChAdOx1-85A and other viral vectors as a BCG-booster vaccination strategy.

Paper-Based Biosensor for the Detection of Sepsis Using MMP-9 Biomarker in FIP Mice Model.

Sepsis is an immune response to a microbial invasion that causes organ injury and dysfunction due to a systemic inflammatory response. Sepsis is a serious, life-threatening condition and a widely recognized global health challenge. Given its high death rate, it is critical to diagnose sepsis and start treatment as early as possible. There is an urgent need for a sensitive and rapid screening method for detecting sepsis. In this study, we investigated the use of MMP-9 as a biomarker for sepsis. A colorimetric paper-based biosensor was used for the detection of MMP-9 utilizing peptide-magnetic nanoparticle conjugates. The method is based on the cleavage of the MMP-9-specific peptide by the protease leading to the detaching of the magnetic beads from the sensor surface and changing of color. A fecal intraperitoneal (FIP) challenge was used to induce sepsis in mice, and an MMP-9 secretion was measured by taking blood and Bronchoalveolar Lavage (BAL) fluid samples at 1 h, 2 h, 4 h, and 20 h (early sepsis) post-challenge intervals. The results of the paper-based sensor for the detection of MMP-9 levels in blood samples and BAL samples were compared with ELISA and Western Blot. We found that both blood and BAL levels of MMP-9 increased immediately and could be detected as early as 1 h in FIP mice post-challenge. Our work adds evidence to the assertion that MMP-9 is a reliable biomarker for the detection of sepsis at early stages.

Repetitive vaccination with intravenous BCG is safe and immunogenic in naïve and SIV-infected macaques

Abstract Tuberculosis (TB) is the leading cause of death in people with HIV (PWH) on or off antiretroviral therapy (ART); thus, new TB vaccine candidates should be evaluated in the setting of HIV. Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the live attenuated bacterial TB vaccine given intradermally (ID) at birth, is contraindicated in PWH. We recently demonstrated that intravenous (IV) delivery of BCG elicits superior immunity and protection compared to ID delivery against Mycobacterium tuberculosis (Mtb) challenge in rhesus macaques. To assess IV BCG in an animal model of HIV infection, we immunized SIVmac251-infected macaques on suppresive ART (6m) and uninfected controls. Rhesus macaques received three doses of IV BCG, 8wks apart, and were monitored for the presence of BCG colony forming units (CFU) in peripheral blood and bronchoalveolar lavage (BAL) and Mtb-specific T cell responses in BAL. Repetitive IV BCG immunization was well-tolerated in SIV-infected and uninfected animals, with no difference in clinical parameters or BCG CFU in blood or BAL. T cell responses after IV BCG were similar, with both groups displaying a 10–50 fold increase in BAL T cell abundance after initial dose. Mtb-specific CD4 and CD8 responses peaked after the initial IV BCG dose and were maintained by subsequent immunizations for at least 6m. These data indicate that IV BCG is safe and immunogenic in SIV-infected, ART-suppressed macaques and that IV BCG boosting maintains T cell responses at the site of infection. NIH Intramural Program

Co-Expression Analysis of Airway Epithelial Transcriptome in Asthma Patients with Eosinophilic vs. Non-Eosinophilic Airway Infiltration.

Asthma heterogeneity complicates the search for targeted treatment against airway inflammation and remodeling. We sought to investigate relations between eosinophilic inflammation, a phenotypic feature frequent in severe asthma, bronchial epithelial transcriptome, and functional and structural measures of airway remodeling. We compared epithelial gene expression, spirometry, airway cross-sectional geometry (computed tomography), reticular basement membrane thickness (histology), and blood and bronchoalveolar lavage (BAL) cytokines of n = 40 moderate to severe eosinophilic (EA) and non-eosinophilic asthma (NEA) patients distinguished by BAL eosinophilia. EA patients showed a similar extent of airway remodeling as NEA but had an increased expression of genes involved in the immune response and inflammation (e.g., KIR3DS1), reactive oxygen species generation (GYS2, ATPIF1), cell activation and proliferation (ANK3), cargo transporting (RAB4B, CPLX2), and tissue remodeling (FBLN1, SOX14, GSN), and a lower expression of genes involved in epithelial integrity (e.g., GJB1) and histone acetylation (SIN3A). Genes co-expressed in EA were involved in antiviral responses (e.g., ATP1B1), cell migration (EPS8L1, STOML3), cell adhesion (RAPH1), epithelial-mesenchymal transition (ASB3), and airway hyperreactivity and remodeling (FBN3, RECK), and several were linked to asthma in genome- (e.g., MRPL14, ASB3) or epigenome-wide association studies (CLC, GPI, SSCRB4, STRN4). Signaling pathways inferred from the co-expression pattern were associated with airway remodeling (e.g., TGF-β/Smad2/3, E2F/Rb, and Wnt/β-catenin).

Further evidence of a type 2 inflammatory signature in chronic obstructive pulmonary disease or emphysema

BackgroundThere is increasing recognition of a type 2 (T2) inflammatory pattern in a subset of patients with chronic obstructive pulmonary disease (COPD) or emphysema, characterized by blood and airway eosinophilia. The mechanism underlying this is not well established. The recognition that CD125 (interleukin [IL]-5 receptor alpha) is expressed on some lung neutrophils and eosinophils in patients with asthma led us to speculate that CD125 may also be expressed on lung neutrophils in patients with COPD or emphysema. ObjectiveTo interrogate the expression of CD125 on lung neutrophils (and, when present, eosinophils) in patients with COPD/emphysema and identify a meaningful biomarker to predict neutrophil CD125 expression, including other markers of T2 inflammation. MethodsWe obtained blood and bronchoalveolar lavage (BAL) samples from patients with physician-diagnosed COPD/emphysema undergoing a clinically indicated bronchoscopy. ResultsWe found that a highly variable percentage of BAL neutrophils indeed expressed surface CD125 (0%-78.7%), with obvious clustering of CD125high and CD125low patterns. No correlation was found with clinical characteristics, blood or BAL eosinophil or neutrophil counts, BAL cytokines, or BAL eosinophil CD125 expression. ConclusionWe conclude that, similar to asthma, lung neutrophils from patients with COPD display interleukin-5 receptor alpha (CD125) on their surface. This along with the frequent presence of IL-4 and IL-5 in airway fluid further suggests a possible role of the T2 pathway in contributing to COPD severity. Trial RegistrationClinicalTrials.gov Identifier: NCT03984799

Supplementation of syringic acid-rich Phrynium pubinerve leaves imparts protection against allergic inflammatory responses by downregulating iNOS, COX-2, and NF-κB expressions

BackgroundThe present study was designed to characterize the role of ethanolic leaf extract of Phrynium pubinerve Blume (EPP) supplement in attenuating allergic inflammation, encouraged by the presence of syringic acid in it, as this phenolic acid is reportedly promising in suppressing serum immunoglobulin E (IgE) and inflammatory cytokine levels. Materials and methodsHPLC-DAD dereplication analysis was performed to determine the presence of the vital polyphenolic metabolites. The efficacy of EPP against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells was evaluated by measuring its inhibitory effects on NO and ROS/RNS production. The expressions of major inflammation-associated molecules (iNOS, COX-2, NF-κB, IL-6, and TNF-α) in RAW 264.7 cells were assessed through Western blot. Physiological and behavioral changes, BMI, and different biochemical parameters in mice blood serum were investigated in the toxicological assays. Formaldehyde-induced paw edema test in mice was conducted using established animal model. TDI-induced allergic model in mice was carried out to determine different allergy-like symptoms, and differential white blood cell (WBC) counts in blood and bronchoalveolar lavage (BAL) fluid. The intermolecular interaction analysis of the identified major metabolite of EPP with H1R and iNOS was studied by molecular docking. ResultsHPLC-DAD analysis showed the presence of syringic acid (89.19 mg/100 g EPP) and a few other compounds. LPS-induced NO generation was reduced by EPP in a concentration-dependent manner, showing IC50 of 28.20 ± 0.27 μg/mL. EPP exhibited a similar inhibitory effect on ROS/RNS production with IC50 of 29.47 ± 2.19 μg/mL. Western blotting revealed that EPP significantly downregulated the expressions of iNOS, COX-2, NF-κB, IL-6, and TNF-α in RAW 264.7 cells when challenged with LPS. The toxicological assays confirmed the dosage and organ-specific safety of EPP. In the formaldehyde-induced paw edema test, EPP caused a 66.41% reduction in mice paw volume at 500 mg/kg dose. It ameliorated TDI-induced allergy-like symptoms and decreased different inflammatory WBCs in mice's blood and BAL fluid in a dose-dependent manner. Finally, syringic acid demonstrated mentionable intermolecular binding affinity towards H1R (−6.6 Kcal/moL) and iNOS (−6.7 Kcal/moL). ConclusionsCollectively, considerable scientific reasoning was obtained in favor of the suppressive potential of EPP against allergic inflammatory responses that are proposed to be exerted via the downregulation of iNOS, COX-2, and NF-κB expressions, H1R antagonism and suppression of cytokines, such as IL-6, and TNF-α.

Effects of early dexamethasone treatment on several markers of inflammation and fibrosis in an animal model of lung silicosis in rats – A pilot study

Abstract Lung silicosis is primarily caused by inhalation of particles of silicon oxide (silica). Despite a huge progress in understanding the interactions among the pathomechanisms of lung silicosis in the last years, there is a lack of effective therapy. With respect to a wide therapeutic action of corticosteroids, the purpose of this pilot study was to evaluate early effects of dexamethasone on several markers of inflammation and lung fibrosis in a rat model of silicosis. The silicosis model was induced by a single transoral intratracheal instillation of silica (50 mg/ml/animal), while the controls received an equivalent volume of sterile saline. The treatment with intraperitoneal dexamethasone initiated the next day after the silica instillation and was given 2-times a week at a dose of 1 mg/kg, while the controls received an equivalent volume of saline. The animals were euthanized 14 or 28 days after the treatment onset. Total and differential counts of leukocytes in the blood and bronchoalveolar lavage (BAL) fluid were determined. The presence of collagen in the bronchioles and lung vessels was detected by Sirius red staining and a smooth muscle mass was detected by smooth muscle actin. In comparison to saline, the instillation of silica increased the total count of circulating leukocytes after 14 and 28 days of the experiment (both p<0.05), which was associated with higher counts of lymphocytes (p<0.05 after 14 days, p>0.05 after 28 days) and slight but non-significant increases in neutrophils and eosinophils (both p>0.05). Although the total cell count in the BAL fluid did not change significantly, the percentages and absolute counts of neutrophils, eosinophils, and lymphocytes (p<0.05, p<0.01 or p<0.001) elevated after 14 and 28 days of the experiment. Silica induced an accumulation of collagen in the bronchioles (p<0.001 after both 14 and 28 days) and pulmonary vessels (p<0.01 after both 14 and 28 days) and elevated a formation of smooth muscle mass (p<0.05 after 14 days, p<0.01 or p<0.001 after 28 days). Treatment with dexamethasone decreased circulating leukocytes (p<0.01) and lymphocytes (p<0.001) and increased neutrophils (p<0.05), which was associated with a slightly decreased total cell count in BAL (p>0.05), decline in lymphocytes (p<0.01), and slight decreases in neutrophils and eosinophils after 28 days of the treatment. Moreover, dexamethasone reduced the accumulation of collagen (p<0.01 after 14 days and p<0.001 after 28 days) and the formation of smooth muscle mass (p<0.01 for bronchioles and p>0.05 for vessels after 24 days, p<0.001 for both bronchioles and vessels after 28 days). In conclusion, early dexamethasone treatment mitigated silica-induced granulocytic-lymphocytic inflammation and decreased a formation of collagen and smooth muscle mass in the bronchiolar and vascular walls, demonstrating a therapeutic potential of dexamethasone in the lung silicosis.

IMPACT OF ALLERGEN SENSITIZATION ON AIRFLOW LIMITATION IN CHILDREN WITH SEVERE ASTHMA

<h3>Introduction</h3> Airflow limitation (AFL) has been shown to predict poor asthma outcomes across maturation. The objective of our study is to identify significant predictors of AFL in children with severe asthma from school-age to adolescence. <h3>Methods</h3> Children ages 5-18 years with treatment-refractory asthma underwent spirometry and diagnostic bronchoscopy. AFL was defined as pre-bronchodilator FEV₁/FVC <0.85 or <90% predicted. Demographics, treatments, and blood and lung markers of inflammation were analyzed with between group non-parametric tests and logistic regression models. <h3>Results</h3> 226 children were included in this analysis. Nearly one-half (n=105, 46%) had AFL. There was no difference in age, gender, BMI, or age of symptom onset between groups. Compared to children without AFL, those with AFL were more likely to have previous ICU admission for asthma, treatment with higher-dose inhaled corticosteroids (ICS), and reported black race. Children with AFL had significantly higher total blood and bronchoalveolar lavage (BAL) eosinophil count, total IgE, and greater number of positive allergen specific serum IgE. In younger children, those with sensitization to ≥4 allergens had greater airflow limitation but the opposite relationship was seen in adolescent children. Similarly, the effects of higher total IgE (>130 IU/mL) on airflow limitation were most prominent in younger children. <h3>Conclusion</h3> Significant predictors of airflow limitation in children with severe asthma include black race, previous ICU admission for asthma, higher-dose ICS treatment, and increased absolute blood and BAL eosinophil count. AFL is driven in part by allergic sensitization in the younger children whereas in older children this associated more with the absence of allergic sensitization.

Reduced frequencies of Foxp3+GARP+ regulatory T cells in COPD patients are associated with multi-organ loss of tissue phenotype

BackgroundExpression of glycoprotein A dominant repeat (GARP) has been reported to occur only in activated human naturally occurring regulatory T cells (Tregs) and their clones, and not in activated effector T cells, indicating that GARP is a marker for bona fide Tregs. A different phenotype of chronic obstructive pulmonary disease (COPD) may have a different immunologic mechanism.ObjectiveTo investigate whether the distribution of Tregs defined by GARP is related to the multi-organ loss of tissue phenotype in COPD.MethodsGARP expression on T cells from peripheral blood and bronchoalveolar lavage (BAL) collected from patients with COPD was examined by flow cytometry. The correlation of GARP expression to clinical outcomes and clinical phenotype, including the body mass index, lung function and quantitative computed tomography (CT) scoring of emphysema, was analyzed.ResultsPatients with more baseline emphysema had lower forced expiratory volume, body mass index (BMI), worse functional capacity, and more osteoporosis, thus, resembling the multiple organ loss of tissue (MOLT) phenotype. Peripheral Foxp3+GARP+ Tregs are reduced in COPD patients, and this reduction reversely correlates with quartiles of CT emphysema severity in COPD. Meanwhile, the frequencies of Foxp3+GARP− Tregs, which are characteristic of pro-inflammatory cytokine production, are significantly increased in COPD patients, and correlated with increasing quartiles of CT emphysema severity in COPD. Tregs in BAL show a similar pattern of variation in peripheral blood.ConclusionDecreased GARP expression reflects more advanced disease in MOLT phenotype of COPD. Our results have potential implications for better understanding of the immunological nature of COPD and the pathogenic events leading to lung damage.

Evaluation of toxicological, anti-allergic and neuropharmacological activities of &lt;em&gt;Bridelia stipularis&lt;/em&gt;: &lt;em&gt;In-vivo&lt;/em&gt; and in-silico studies

Bridelia stipularis locally known as ‘Harinhara’ native in hill tract area. The plant is traditionally used in cough, asthma and catarrh by the Chakma ethnic people of Bangladesh. In this study, we investigated the toxicological, anti-allergic, neuropharmacological potential of leaves extract and profiling of its bioactive polyphenols by HPLC for in-silico molecular docking. Phytochemical test confirmed the existence of phytochemicals including tannin, flavonoid, saponin, alkaloid, glycoside etc. In toxicity study, the extract was safe up to 3 g/kg oral dose in mice. The studied showed no organ specific toxicity at 500 mg/kg oral dose. Anti-allergic activity performed by using TDI-induced allergic mice model. The extract significantly reduced the TDI-induced allergy like symptoms such as scratching, sneezing, swelling (P < 0.05). Additionally, the extract also ameliorated the leukocytes count in blood and bronchoalveolar lavage (BAL) fluid. The neuropharmacological activity test was conducted by following the open field method in mice model, at both doses of extract at 300 and 500 mg/kg showed mild CNS depressant effect. HPLC profiling identified the presence of seven phenolic compounds, among these rutin hydrate demonstrated the best docking score of -7.4 kcal/mol same as anti-allergic drug against (H1R). On the other hand exhibited rosmarinic acid and myricetin -7.9 and -7.8 kcal/mol against GABAA whereas standard CNS drug showed -7.7 kcal/mol. In conclusion, B. stipularis is safe and rich in different polyphenolic compounds which were found effective against diseases.

The Cholinergic Drug Galantamine Ameliorates Acute Respiratory Distress Syndrome in Mice

Acute respiratory distress syndrome (ARDS) is a life‐threatening form of acute lung injury and respiratory failure. The pandemic of SARS‐CoV‐2 has resulted in over 5 million deaths worldwide and many of those deaths have been attributed to complications associated with ARDS. No efficient pharmacological treatments for ARDS are currently available. In addition to lung injury and pulmonary inflammation, patients with ARDS have increased systemic inflammation. Previous insights have highlighted the role of cholinergic signalling in the vagus nerve‐based inflammatory reflex in controlling inflammation (Annu Rev Immunol, 2018, 36:783). Cholinergic anti‐inflammatory signalling can be activated by galantamine ‐ a centrally acting acetylcholinesterase inhibitor that is also clinically approved (for the treatment of Alzheimer’s disease). Here we examined the efficacy of galantamine in attenuating ARDS severity and inflammation in mice. We used a recently developed model of direct acute lung injury and ARDS, in which intratracheal (i.t.) hydrochloric acid (0.1N HCl, 2ml/kg) instillation is combined with LPS (10 mg/kg, i.t.) instillation 24h later in anesthetized mice. Male, 12‐week‐old C57BL/6 mice (n=7‐9), were subjected to this two hit ARDS model and treated 30 mins prior to each insult with galantamine (4 mg/kg, i.p.) or vehicle. Mice were euthanized 6h after LPS instillation and blood and bronchoalveolar lavage (BAL) fluid were collected. Galantamine significantly lowered the levels of BAL myeloperoxidase – an indicator of the accumulation of activated neutrophils in the airspaces of the lung (p = 0.0021) and the BAL total protein ‐ an indicator of alveolar permeability (p = 0.0076), compared with vehicle‐treated mice. BAL pro‐inflammatory cytokine levels were also reduced by galantamine, compared with vehicle‐treated mice – TNF (p = 0.0274), IL‐6 (p = 0.0009), and IL‐1β (p = 0.0380). In addition, treatment with galantamine significantly lowered the levels of serum pro‐inflammatory cytokines ‐ TNF (p = 0.0015), IL‐6 (p = 0.0089), and IL‐1β (p = 0.0149). In conclusion, galantamine reduced the severity of ARDS in mice and alleviated the local and systemic inflammatory responses. These results are of interest for further preclinical and clinical development of galantamine in the treatment of ARDS.

Excess iron promotes emergence of foamy macrophages that overexpress ferritin in the lungs of silicosis patients.

Background and objectiveInhalation of high concentrations of respirable crystalline silica (RCS) can lead to silicosis. RCS contains varying levels of iron, which can cause oxidative stress and stimulate ferritin production. This study evaluated iron‐related and inflammatory markers in control and silicosis patients.MethodsA cohort of stone benchtop industry workers (n = 18) were radiologically classified by disease severity into simple or complicated silicosis. Peripheral blood and bronchoalveolar lavage (BAL) were collected to measure iron, ferritin, C‐reactive protein, serum amyloid A and serum silicon levels. Ferritin subunit expression in BAL and transbronchial biopsies was analysed by reverse transcription quantitative PCR. Lipid accumulation in BAL macrophages was assessed by Oil Red O staining.ResultsSerum iron levels were significantly elevated in patients with silicosis, with a strong positive association with serum ferritin levels. In contrast, markers of systemic inflammation were not increased in silicosis patients. Serum silicon levels were significantly elevated in complicated disease. BAL macrophages from silicosis patients were morphologically consistent with lipid‐laden foamy macrophages. Ferritin light chain (FTL) mRNA expression in BAL macrophages was also significantly elevated in simple silicosis patients and correlated with systemic ferritin.ConclusionOur findings suggest that elevated iron levels during the early phases of silicosis increase FTL expression in BAL macrophages, which drives elevated BAL and serum ferritin levels. Excess iron and ferritin were also associated with the emergence of a foamy BAL macrophage phenotype. Ferritin may represent an early disease marker for silicosis, where increased levels are independent of inflammation and may contribute to fibrotic lung remodelling.

Quantitative Determination of Unbound Piperacillin and Imipenem in Biological Material from Critically Ill Using Thin-Film Microextraction-Liquid Chromatography-Mass Spectrometry.

β-Lactam antibiotics are most commonly used in the critically ill, but their effective dosing is challenging and may result in sub-therapeutic concentrations that can lead to therapy failure and even promote antimicrobial resistance. In this study, we present the analytical tool enabling specific and sensitive determination of the sole biologically active fraction of piperacillin and imipenem in biological material from the critically ill. Thin-film microextraction sampling technique, followed by rapid liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, was optimized and validated for the quantitative determination of antibiotics in blood and bronchoalveolar lavage (BAL) specimens collected from intensive care unit (ICU) patients suffering from ventilation-associated pneumonia (n = 18 and n = 9, respectively). The method was optimized and proved to meet the criteria of US Food and Drug Administration (FDA) guidelines for bioanalytical method validation. Highly selective, sensitive, accurate and precise analysis by means of thin-film microextraction–LC-MS/MS, which is not affected by matrix-related factors, was successfully applied in clinical settings, revealing poor penetration of piperacillin and imipenem from blood into BAL fluid (reflecting the site of bacterial infection), nonlinearity in antibiotic binding to plasma-proteins and drug-specific dependence on creatinine clearance. This work demonstrates that only a small fraction of biologically active antibiotics reach the site of infection, providing clinicians with a high-throughput analytical tool for future studies on personalized therapeutic drug monitoring when tailoring the dosing strategy to an individual patient.

Peroxisome proliferator-activated receptor gamma gene variants modify human airway and systemic responses to indoor dibutyl phthalate exposure

BackgroundSingle nucleotide polymorphisms (SNPs) of peroxisome proliferator-activated receptor gamma (PPAR-γ; gene: PPARG) and oxidative stress genes are associated with asthma risk. However, whether such variants modulate responses to dibutyl phthalate (DBP), a common plasticizer associated with increased asthma development, remains unknown. The purpose of this study is to investigate how SNPs in PPARG and oxidative stress genes, as represented by two separate genetic risk scores, modify the impact of DBP exposure on lung function and the airway and systemic response after an inhaled allergen challenge.MethodsWe conducted a double-blinded human crossover study with sixteen allergen-sensitized participants exposed for three hours to DBP and control air on distinct occasions separated by a 4-week washout. Each exposure was followed by an allergen inhalation challenge; subsequently, lung function was measured, and blood and bronchoalveolar lavage (BAL) were collected and analyzed for cell counts and allergen-specific immunoglobulin E (IgE). Genetic risk scores for PPAR-γ (P-GRS; weighted sum of PPARG SNPs rs10865710, rs709158, and rs3856806) and oxidative stress (OS-GRS; unweighted sum of 16 SNPs across multiple genes) were developed, and their ability to modify DBP effects were assessed using linear mixed-effects models.ResultsP-GRS and OS-GRS modified DBP effects on allergen-specific IgE in blood at 20 h (interaction effect [95% CI]: 1.43 [1.13 to 1.80], p = 0.005) and 3 h (0.99 [0.98 to 1], p = 0.03), respectively. P-GRS also modified DBP effects on Th2 cells in blood at 3 h (− 25.2 [− 47.7 to − 2.70], p = 0.03) and 20 h (− 39.1 [− 57.9 to − 20.3], p = 0.0005), and Th2 cells in BAL at 24 h (− 4.99 [− 8.97 to − 1.01], p = 0.02). An increasing P-GRS associated with reduced DBP effect on Th2 cells. Neither GRS significantly modified DBP effects on lung function parameters.ConclusionsPPAR-γ variants modulated several airway and systemic immune responses to the ubiquitous chemical plasticizer DBP. Our results suggest that PPAR-γ variants may play a greater role than those in oxidative stress-related genes in airway allergic responses to DBP.Trial registration: This study reports results from The Phthalate-Allergen Immune Response Study that was registered on ClinicalTrials.gov with identification NCT02688478.

Investigation of Anti-hyperglycemic, Anti-allergic activities of ethanolic extract from &lt;em&gt;Brownlowia tersa&lt;/em&gt; (L.) stem

Brownlowia tersa (L.) (Family: Malvaceae), has been used traditionally to treat inflammation, pain, diarrhea, allergic reaction etc. The aim of the recent study is to investigate the ethanolic extract of Brownlowia tersa (L.) stem for its anti-hyperglycemic and anti-allergic activity as well as to identify the phytochemicals constituents. Blood glucose level in mice, in anti-hyperglycemic activity, evaluated by OGTT (oral glucose tolerance test) considerably reduced by the extract at both dose 250 and 500 mg/kg b. wt. The extract also decreased significantly the allergic-like symptoms like sneezing, scratching, and nasal score induced by TDI in mice model. The count of WBC (total and differential) in both blood and Bronchoalveolar lavage (BAL) fluid were also reduced in the extract treated group. In qualitative phytochemical investigation, existence of different bioactive phytochemicals like alkaloids, flavonoids, steroids and tannins were observed. So the investigation suggests the anti-hyperglycemic and anti-allergic activity of B. tersa stem extract due to the availability of one or more active metabolites in it.

Abstract 11119: Real-Time Nanopore Sequencing for Bacterial Pneumonia After Out-of-Hospital Cardiac Arrest, a Pilot Proof-of-Concept Study

Introduction: Pneumonia is common after OHCA but is difficult to diagnose in the first 72 hours following ROSC, this results in early untargeted antibiotic administration based on non-specific imaging and laboratory findings. Antibiotic resistance is rising, is influenced by untargeted antibiotic administration, and can increase patient morbidity and mortality as well as healthcare costs. Precision methods of bacterial pathogen detection in OHCA patients are needed to improve patient care. This proof-of-concept pilot study aimed to assess feasibility of bacterial pathogen sequencing and comparability of sequencing results to clinical culture after OHCA. Methods: Blood and bronchoalveolar lavage (BAL) were obtained from residual clinical specimens collected within 12 hours of ROSC. Bacterial DNA was extracted using the Qiagen PowerLyzer PowerSoil DNA kit, sequenced using the MinION nanopore sequencer, and analyzed with Oxford Nanopore Technologies’ EPI2ME bioinformatics software. Sequencing results were compared to culture results using McNemar’s chi-square statistic. Study-defined pneumonia was based on presence of at least two characteristics within 72 hours of ROSC: fever (temperature ≥38°C); persistent leukocytosis &gt;15,000 or leukopenia &lt;3,500 for 48 hours; persistent chest radiography infiltrates for 48 hours per clinical radiology read; bacterial pathogen cultured. Results: We enrolled 38 consecutive OHCA subjects: mean age 61.8 years (18.0); 16 (42%) female; 25 (66%) White, 7 (18%) Black, 6 (16%) “Other” race; 7 subjects (18%) survived and 31 (82%) died; 16 (42%) subjects had pneumonia. Sequencing results were available in 12 hours while culture results were available in 48-72 hours after collection. There was a non-significant difference in the proportion of the same pathogens identified for each method per McNemar’s chi-square: p = 0.38, difference of 0.095 (-0.095, 0.286). Conclusions: Nanopore sequencing detects pathogenic bacteria comparable to clinical microbiologic culture and in less time. This technology can produce a paradigm shift in early bacterial pathogen detection in OHCA survivors, which can improve patient care. The technology is applicable to other patient populations and for viral and fungal pathogens.

Humoral Immune Status in Relation to Outcomes in Patients with Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease, in which inflammation is thought to only play a secondary role. Several factors associated with acute exacerbations of IPF (AE-IPF) have been identified, including infections. This study investigated whether humoral immunodeficiency or increased inflammatory markers at diagnosis were associated with AE-IPF and survival. Four-hundred-and-nine patients diagnosed with IPF between 2011 and 2017 were retrospectively included. Immune status investigations at diagnosis included measurement of serum immunoglobulins (available in 38%), leukocyte and lymphocyte subsets in blood and bronchoalveolar lavage (BAL) fluid (available in 58%), as well as response to pneumococcal vaccination (available in 64%). Serum immunoglobulins or IgG subclass levels were below the lower limit of normal in 6%. The response to pneumococcal vaccination was severely impaired in 1%. Thirteen percent of patients developed an AE-IPF (4.7% per year). AE-IPF were associated with elevated lymphocytes in BAL fluid at diagnosis (p = 0.03). Higher serum IgA and IgG at diagnosis were associated with worse survival (p = 0.01; and p = 0.04), as were an increased BAL lymphocyte percentage (p = 0.005), and higher blood leukocytes and neutrophils (p = 0.01; and p = 0.0005). In a multivariate model, only BAL lymphocyte count retained statistical significance (p = 0.007). The prevalence of humoral immunodeficiencies was low in patients with IPF and not associated with AE-IPF or survival. Elevated lymphocytes in BAL were associated with the development of AE-IPF and worse survival. Higher serum immunoglobulins and immune cells in blood were also associated with worse survival. The local immune response in the lungs may be a target for future therapies.

LOX-1-Expressing Immature Neutrophils Identify Critically-Ill COVID-19 Patients at Risk of Thrombotic Complications.

BackgroundLymphopenia and the neutrophil/lymphocyte ratio may have prognostic value in COVID-19 severity.ObjectiveWe investigated neutrophil subsets and functions in blood and bronchoalveolar lavage (BAL) of COVID-19 patients on the basis of patients’ clinical characteristics.MethodsWe used a multiparametric cytometry profiling based to mature and immature neutrophil markers in 146 critical or severe COVID-19 patients.ResultsThe Discovery study (38 patients, first pandemic wave) showed that 80% of Intensive Care Unit (ICU) patients develop strong myelemia with CD10−CD64+ immature neutrophils (ImNs). Cellular profiling revealed three distinct neutrophil subsets expressing either the lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), the interleukin-3 receptor alpha (CD123), or programmed death-ligand 1 (PD-L1) overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1- or CD123-expressing ImNs is positively correlated with clinical severity, cytokine storm (IL-1β, IL-6, IL-8, TNFα), acute respiratory distress syndrome (ARDS), and thrombosis. BALs of patients with ARDS were highly enriched in LOX-1-expressing ImN subsets and in antimicrobial neutrophil factors. A validation study (118 patients, second pandemic wave) confirmed and strengthened the association of the proportion of ImN subsets with disease severity, invasive ventilation, and death. Only high proportions of LOX-1-expressing ImNs remained strongly associated with a high risk of severe thrombosis independently of the plasma antimicrobial neutrophil factors, suggesting an independent association of ImN markers with their functions.ConclusionLOX-1-expressing ImNs may help identifying COVID-19 patients at high risk of severity and thrombosis complications.

Comprehensive Immunologic Evaluation of Bronchoalveolar Lavage Samples from Human Patients with Moderate and Severe Seasonal Influenza and Severe COVID-19.

Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or seasonal influenza may lead to respiratory failure requiring intubation and mechanical ventilation. The pathophysiology of this respiratory failure is attributed to local immune dysregulation, but how the immune response to viral infection in the lower airways of the human lung differs between individuals with respiratory failure and those without is not well understood. We used quantitative multiparameter flow cytometry and multiplex cytokine assays to evaluate matched blood and bronchoalveolar lavage (BAL) samples from control human subjects, subjects with symptomatic seasonal influenza who did not have respiratory failure, and subjects with severe seasonal influenza or SARS-CoV-2 infection with respiratory failure. We find that severe cases are associated with an influx of nonclassical monocytes, activated T cells, and plasmablast B cells into the lower airways. Cytokine concentrations were not elevated in the lower airways of moderate influenza patients compared with controls; however, 28 of 35 measured cytokines were significantly elevated in severe influenza, severe SARS-CoV-2 infection, or both. We noted the largest elevations in IL-6, IP-10, MCP-1, and IL-8. IL-1 family cytokines and RANTES were higher in severe influenza infection than severe SARS-CoV-2 infection. Interestingly, only the concentration of IP-10-correlated between blood and BAL during severe infection. Our results demonstrate inflammatory immune dysregulation in the lower airways during severe viral pneumonia that is distinct from lower airway responses seen in human patients with symptomatic, but not severe, illness and suggest that measurement of blood IP-10 concentration may predict this unique dysregulation.

Pulmonary and blood dendritic cells from sarcoidosis patients more potently induce IFNγ-producing Th1 cells compared with monocytes.

Sarcoidosis is a systemic inflammatory disease mainly affecting the lungs. The hallmark of sarcoidosis are granulomas that are surrounded by activated T cells, likely targeting the disease-inducing antigen. IFNγ-producing Th1 and Th17.1 T cells are elevated in sarcoidosis and associate with disease progression. Monocytes and dendritic cells (DCs) are antigen-presenting cells (APCs) and required for T cell activation. Several subsets of monocytes and DCs with different functions were identified in sarcoidosis. However, to what extent different monocyte and DC subsets can support activation and skewing of T cells in sarcoidosis is still unclear. In this study, we performed a transcriptional and functional side-by-side comparison of sorted monocytes and DCs from matched blood and bronchoalveolar lavage (BAL) fluid of sarcoidosis patients. Transcriptomic analysis of all subsets showed upregulation of genes related to T cell activation and antigen presentation in DCs compared with monocytes. Allogeneic T cell proliferation was higher after coculture with monocytes and DCs from blood compared with BAL and DCs induced more T cell proliferation compared with monocytes. After coculture, proliferating T cells showed high expression of the transcription factor Tbet and IFNγ production. We also identified Tbet and RORγt coexpressing T cells that mainly produced IFNγ. Our data show that DCs rather than monocytes from sarcoidosis patients have the ability to activate and polarize T cells towards Th1 and Th17.1 cells. This study provides a useful in vitro tool to better understand the contribution of monocytes and DCs to T cell activation and immunopathology in sarcoidosis.