Bronchoalveolar Lavage Lymphocytes Research Articles

Self-report underestimates the frequency of the acute respiratory exacerbations of COPD but is associated with BAL neutrophilia and lymphocytosis: an observational study

RationaleResearch studies typically quantify acute respiratory exacerbation episodes (AECOPD) among people with chronic obstructive pulmonary disease (COPD) based on self-report elicited by survey questionnaire. However, AECOPD quantification by self-report could be inaccurate, potentially rendering it an imprecise tool for identification of those with exacerbation tendency.ObjectiveDetermine the agreement between self-reported and health records-documented quantification of AECOPD and their association with airway inflammation.MethodsWe administered a questionnaire to elicit the incidence and severity of respiratory exacerbations in the three years preceding the survey among current or former heavy smokers with or without diagnosis of COPD. We then examined electronic health records (EHR) of those with COPD and those without (tobacco-exposed persons with preserved spirometry or TEPS) to determine whether the documentation of the three-year incidence of moderate to very severe respiratory exacerbations was consistent with self-report using Kappa Interrater statistic. A subgroup of participants also underwent bronchoalveolar lavage (BAL) to quantify their airway inflammatory cells. We further used multivariable regressions analysis to estimate the association between respiratory exacerbations and BAL inflammatory cell composition with adjustment for covariates including age, sex, height, weight, smoking status (current versus former) and burden (pack-years).ResultsOverall, a total of 511 participants completed the questionnaire, from whom 487 had EHR available for review. Among the 222 participants with COPD (70 ± 7 years-old; 96% male; 70 ± 38 pack-years smoking; 42% current smoking), 57 (26%) reported having any moderate to very severe AECOPD (m/s-AECOPD) while 66 (30%) had EHR documentation of m/s-AECOPD. However, 42% of those with EHR-identified m/s-AECOPD had none by self-report, and 33% of those who reported m/s-AECOPD had none by EHR, suggesting only moderate agreement (Cohen’s Kappa = 0.47 ± 0.07; P < 0.001). Nevertheless, self-reported and EHR-identified m/s-AECOPD events were both associated with higher BAL neutrophils (ß ± SEM: 3.0 ± 1.1 and 1.3 ± 0.5 per 10% neutrophil increase; P ≤ 0.018) and lymphocytes (0.9 ± 0.4 and 0.7 ± 0.3 per 10% lymphocyte increase; P ≤ 0.041). Exacerbation by either measure combined was associated with a larger estimated effect (3.7 ± 1.2 and 1.0 ± 0.5 per 10% increase in neutrophils and lymphocytes, respectively) but was not statistically significantly different compared to the self-report only approach. Among the 184 TEPS participants, there were fewer moderate to very severe respiratory exacerbations by self-report (n = 15 or 8%) or EHR-documentation (n = 9 or 5%), but a similar level of agreement as those with COPD was observed (Cohen’s Kappa = 0.38 ± 0.07; P < 0.001).DiscussionWhile there is modest agreement between self-reported and EHR-identified m/s-AECOPD, events are missed by relying on either method alone. However, m/s-AECOPD quantified by self-report or health records is associated with BAL neutrophilia and lymphocytosis.

Dimethyl sulfoxide as a novel therapy in a murine model of acute lung injury.

The endothelial glycocalyx on the luminal surface of endothelial cells contributes to the permeability barrier of the pulmonary vasculature. Dimethyl sulfoxide (DMSO) has a disordering effect on plasma membranes, which prevents the formation of ordered membrane domains important in the shedding of the endothelial glycocalyx. We hypothesized that DMSO would protect against protein leak by preserving the endothelial glycocalyx in a murine model of acute respiratory distress syndrome (ARDS). C57BL/6 mice were given ARDS via intratracheally administered lipopolysaccharide (LPS). Dimethyl sulfoxide (220 mg/kg) was administered intravenously for 4 days. Animals were sacrificed postinjury day 4 after bronchoalveolar lavage (BAL). Bronchoalveolar lavage cell counts and protein content were quantified. Lung sections were stained with fluorescein isothiocyanate-labeled wheat germ agglutinin to quantify the endothelial glycocalyx. Human umbilical vein endothelial cells (HUVECs) were exposed to LPS. Endothelial glycocalyx was measured using fluorescein isothiocyanate-labeled wheat germ agglutinin, and co-immunoprecipitation was performed to measure interaction between sheddases and syndecan-1. Dimethyl sulfoxide treatment resulted in greater endothelial glycocalyx staining intensity in the lung when compared with sham (9,641 vs. 36,659 arbitrary units, p < 0.001). Total BAL cell counts were less for animals receiving DMSO (6.93 × 10 6 vs. 2.49 × 10 6 cells, p = 0.04). The treated group had less BAL macrophages (189.2 vs. 76.9 cells, p = 0.02) and lymphocytes (527.7 vs. 200.0 cells, p = 0.02). Interleukin-6 levels were lower in DMSO treated. Animals that received DMSO had less protein leak in BAL (1.48 vs. 1.08 μg/μL, p = 0.02). Dimethyl sulfoxide prevented LPS-induced endothelial glycocalyx loss in HUVECs and reduced the interaction between matrix metalloproteinase 16 and syndecan-1. Systemically administered DMSO protects the endothelial glycocalyx in the pulmonary vasculature, mitigating pulmonary capillary leak after acute lung injury. Dimethyl sulfoxide also results in decreased inflammatory response. Dimethyl sulfoxide reduced the interaction between matrix metalloproteinase 16 and syndecan-1 and prevented LPS-induced glycocalyx damage in HUVECs. Dimethyl sulfoxide may be a novel therapeutic for ARDS.

The efficacy of N-acetylcysteine in decreasing airway inflammation and mucus accumulation in horses with 18 hours of head confinement.

During transportation many horses develop post-transportation infection, which can be life-threatening and end their sport career. Preventing mucus accumulation and inflammation during transportation is vital, emphasizing the need for effective strategies to enhance overall horse health welfare. Assess the impact of N-acetylcysteine (NAC) on mucus accumulation and inflammation in horses subjected to 18 hours of head confinement. Six healthy crossbred horses, 5.3 ± 2.1 years of age and weighing 387 ± 30 kg. Prospective placebo-controlled cross-over design study. The horses' heads were restrained in their stalls for a period of 18 hours. They were studied under 4 conditions: Not confined (NC): before head confinement, placebo (P), and confined head (CH): 18 hours of head confinement without treatment, and N-Acetylcysteine (NAC): 18 hours of head confinement treated with NAC before confinement (15 mg/kg/day NAC PO for 3 days). Bronchoalveolar lavage (BAL) was performed in each condition. Mucus accumulation along the trachea was evaluated by endoscopy. Endoscopic scores were significantly different between CH and other conditions, whereas no significant differences were found among NC, P, and NAC. The BAL cell count (34 291 ± 2624 cells/μL), neutrophil and lymphocyte count (18 601 ± 3193 cells/μL and 3337.4 ± 593 cells/μL, respectively) in CH were significantly higher compared to NAC. Neutrophil percentage was significantly higher in CH (53.8 ± 8%) compared to horses that received NAC (20.08 ± 8%). Conversely, in comparison to NAC (66.33 ± 9%), the percentage of macrophages was significantly lower in CH (35.7 ± 10%). N-acetylcysteine was found to significantly decrease mucus accumulation and inflammatory cell counts in horses with head confinement.

Dimethyl malonate protects the lung in a murine model of acute respiratory distress syndrome.

Succinate is a proinflammatory citric acid cycle metabolite that accumulates in tissues during pathophysiological states. Oxidation of succinate after ischemia-reperfusion leads to reversal of the electron transport chain and generation of reactive oxygen species. Dimethyl malonate (DMM) is a competitive inhibitor of succinate dehydrogenase, which has been shown to reduce succinate accumulation. We hypothesized that DMM would protect against inflammation in a murine model of ARDS. C57BL/6 mice were given ARDS via 67.7 μg of intratracheally administered lipopolysaccharide. Dimethyl malonate (50 mg/kg) was administered via tail vein injection 30 minutes after injury, then daily for 3 days. The animals were sacrificed on day 4 after bronchoalveolar lavage (BAL). Bronchoalveolar lavage cell counts were performed to examine cellular influx. Supernatant protein was quantified via Bradford protein assay. Animals receiving DMM (n = 8) were compared with those receiving sham injection (n = 8). Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify the endothelial glycocalyx (EGX). Total cell counts in BAL was less for animals receiving DMM (6.93 × 10 6 vs. 2.46 × 10 6 , p = 0.04). The DMM group had less BAL macrophages (168.6 vs. 85.1, p = 0.04) and lymphocytes (527.7 vs. 248.3; p = 0.04). Dimethyl malonate-treated animals had less protein leak in BAL than sham treated (1.48 vs. 1.15 μg/μl, p = 0.03). Treatment with DMM resulted in greater staining intensity of the EGX in the lung when compared with sham (12,016 vs. 15,186 arbitrary units, p = 0.03). Untreated animals had a greater degree of weight loss than treated animals (3.7% vs. 1.1%, p = 0.04). Dimethyl malonate prevented the upregulation of monocyte chemoattractant protein-1 (1.66 vs. 0.92 RE, p = 0.02) and ICAM-1 (1.40 vs. 1.01 RE, p = 0.05). Dimethyl malonate reduces lung inflammation and capillary leak in ARDS. This may be mediated by protection of the EGX and inhibition of monocyte chemoattractant protein-1 and ICAM-1. Dimethyl malonate may be a novel therapeutic for ARDS.

Evaluation of PD-1 T lymphocytes in bronchoalveolar lavage in lung cancer compared to benign lung diseases

The expression of the programmed cell death protein 1 (PD-1) has been shown to be markedly increased in tumor-infiltrating lymphocytes. However, the proportion of PD-1 + T cells in the bronchoalveolar lavage (BAL) of lung cancer patients has not been sufficiently evaluated so far. In this prospective study, the proportion of PD-1 + CD4 + as well as PD-1 + CD8 + T cells in BAL samples, isolated from patients with lung cancer, asthma or interstitial lung disease (ILD), were determined via flow cytometry and compared for differences. Bronchoalveolar lavage was performed in 34 patients (14 patients with lung cancer, 10 patients with asthma, 10 patients with ILD). The highest median proportion of PD-1 + CD4 + or PD-1 + CD8 + T cells were found in patients with ILD (83.1% [IQR 72.1; 87.5] and 73.8% [IQR 60.3; 86.3]) followed by patients with lung cancer (66.4% [IQR 59; 69] and 77.1% [IQR 35.8; 82.3]) and patients with asthma (61.3% [IQR 57.4; 70.5] and 57.3% [IQR 46; 65]). Thereby, the difference in the proportion of PD-1 + CD3 + CD4 + BAL cells between ILD patients and asthmatics was significantly different (p = 0.04). The proportion of PD-1 + CD4 + and PD-1 + CD8 + T cells in the BAL of patients with lung cancer did not differ significantly to patients with benign lung diseases. The highest proportion was observed in ILD patients suggesting further research to evaluate the role of the PD-1/PD-L1 pathway in ILD patients.

Real-Life Diagnostic Performance of the Hypersensitivity Pneumonitis Guidelines: A Multicenter Cohort Study.

Hypersensitivity pneumonitis (HP) is a heterogeneous interstitial lung disease (ILD) that may be difficult to confidently diagnose. Recently, the 2020 ATS/JRS/ALAT HP diagnostic guidelines were published, yet data validating their performance in real-life settings are scarce. We aimed to assess the diagnostic performance of the HP guidelines compared to the gold-standard multidisciplinary discussion (MDD). For this purpose, we included consecutive ILD patients that underwent diagnostic bronchoscopy between 2017 and 2020 in three large medical centers. Four diagnostic factors (antigen exposure history, chest computed tomography pattern, bronchoalveolar lavage lymphocyte count, and histology results) were used to assign guidelines-based HP diagnostic confidence levels for each patient. A sensitivity analysis was performed, with MDD diagnosis as the reference standard. Overall, 213 ILD patients were included, 45 (21%) with an MDD diagnosis of HP. The guidelines' moderate (≥70%) confidence threshold produced optimal performance with 73% sensitivity for HP, 89% specificity, and a J-index of 0.62. The area under the receiver operating characteristic curve (AUC) for a correct guidelines-based diagnosis was 0.86. The guidelines had better performance for non-fibrotic than fibrotic HP (AUC 0.92 vs. 0.82). All diagnostic factors, except bronchoalveolar lavage lymphocyte count, were independent predictors for MDD diagnosis of HP in a multivariate analysis. In conclusion, the HP guidelines exhibited a good diagnostic performance compared to MDD diagnosis in real-life setting.

Triple-tyrosine kinase inhibition by BIBF1000 attenuates airway and pulmonary arterial remodeling following chronic allergen challenges in mice

BackgroundAirway remodeling is an important pathological feature of chronic airway diseases, which leads to a progressive decline in lung function. The present study examined the anti-remodeling and anti- inflammatory effect of BIBF1000, a triple-tyrosine kinase inhibitor that targets VEGF, PDGF, and FGF receptor signaling in a mouse model of repeated ovalbumin (OVA) challenges.MethodsFemale Balb-c mice were immunized intraperitoneally on days 0 and 12 with 50 µg ovalbumin plus 1 mg of Al(OH)3 in 200 μl saline. Intranasal OVA challenges (20 µg/50 µl in PBS) were administered on days 26, 29, and 31, and were repeated twice a week for 3 months. Animals received vehicle or BIBF1000 (25 mg/kg, b.i.d.) through gavage from day 26 to the end of fourth month. On day 120, bronchoalveolar lavage (BAL) and lung tissue were collected for biochemical and immunohistological analysis.ResultsCompared to vehicle controls, treatment with BIBF1000 reduced the numbers of BAL eosinophils, macrophages, neutrophils, and lymphocytes by 70.0%, 57.9%, 47.5%, and 63.0%, respectively, and reduced IL-5 and IL-13 in BAL. Treatment with BIBF1000 reduced airway mucus secretion, peribronchial fibrosis, small airway, and pulmonary arterial wall thickness, compared to vehicle controls. Furthermore, treatment with BIBF1000 also reduced the expression of inflammatory mediators (TNF-α, IL-1β, IL-5, IL-13, MMP-2, MMP-9, COX-2, and iNOS) and inhibited ERK and AKT phosphorylation.ConclusionsThe protective effect afforded by triple-tyrosine kinase inhibition with BIBF1000 in reducing allergen-induced airway and arterial remodeling was associated with down-regulation of inflammatory mediators, as well as inhibition of ERK and AKT signaling pathways.

Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences.

Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and compares to human asthma. The pathogenesis of EA is most likely immune-mediated, yet incompletely understood. To study the immune response in the affected lower airways, mixed leukocytes were acquired through bronchoalveolar lavage (BAL) and the cell populations were analyzed on a single-cell basis by flow cytometry (FC). Samples of 38 horses grouped as respiratory healthy or affected by mild to moderate (mEA) or severe EA (sEA) according to their history, clinical signs, and BAL cytology were analyzed. Using FC, BAL cells and PBMC were comprehensively characterized by cell surface markers ex vivo. An increased percentage of DH24A+ polymorphonuclear cells, and decreased percentages of CD14+ macrophages were detected in BAL from horses with sEA compared to healthy horses or horses with mEA, while lymphocyte proportions were similar between all groups. Independently of EA, macrophages in BAL were CD14+CD16+, which contrasts the majority of CD14+CD16- classical monocytes in PBMC. Percentages of CD16-expressing BAL macrophages were reduced in BAL from horses with sEA compared to healthy horses. While PBMC lymphocytes predominantly contain CD4+ T cells, B cells and few CD8+ T cells, BAL lymphocytes comprised mainly CD8+ T cells, fewer CD4+ T cells and hardly any B cells. These lymphocyte subsets’ distributions were similar between all groups. After PMA/ionomycin stimulation in vitro, lymphocyte activation (CD154 and T helper cell cytokine expression) was analyzed in BAL cells of 26 of the horses and group differences were observed (p=0.01–0.11). Compared to healthy horses’ BAL, CD154+ lymphocytes from horses with mEA, and CD4+IL-17A+ lymphocytes from horses with sEA were increased in frequency. Activated CD4+ T helper cells were more frequent in asthmatics’ (mEA, sEA) compared to healthy horses’ PBMC lymphocytes. In summary, FC analysis of BAL cells identified increased polymorphonuclear cells frequencies in sEA as established, while macrophage percentages were mildly reduced, and lymphocyte populations remained unaffected by EA. Cytokine production differences of BAL lymphocytes from horses with sEA compared to healthy horses’ cells point towards a functional difference, namely increased local type 3 responses in sEA.

Cytopathological Findings in Bronchoalveolar Lavage from Patients with COVID-19

Information on cellular analysis of bronchoalveolar lavage (BAL) in patients with COVID-19 is limited. Some studies have described an increase in lymphocyte percentage or exuberant plasmacytosis. Some reports addressed the importance of molecular testing on BAL samples to confirm COVID-19 pneumonia, in clinically highly suspected patients with consecutive negative nasopharyngeal swab results. In addition to atypical lymphocytes in the peripheral blood, morphologic findings of atypical lymphocytes in BAL were also reported in a few patients. The objective of this study was to describe the cytopathic characteristics identified, any data presented here are descriptives and intended to trigger further research. Three general aspects have been evaluated in each sample: reactive changes, virus-related pathological changes, and differential leukocyte count. Seventeen samples were collected. All samples were negative for malignancy, with an inflammatory background, predominantly lymphohistiocytic in 5 samples, histiocytic in 9, and 3 with predominantly neutrophilic. Hemosiderin-laden macrophages were observed in 12/17. Nonspecific reactive cell changes were identified in 4 samples, including bronchial, alveolar, and reserve cell hyperplasia. Virus-related pathological changes were observed in 14 samples, such as loss of nuclear chromatin pattern, lymphocytes with atypical nuclei, nuclear and cytoplasmic inclusions, multinucleations in bronchial cells and macrophages, or multinucleated giant cells. The identification of multinucleated giant cells could represent a cytopathic effect induced by the virus, at the same time the nuclear clearance of pneumocytes as a possible direct effect. BAL is a procedure aimed at obtaining cells from the respiratory tract that can provide valuable and rapid information. It is important to collect and describe as many cytopathological findings as possible, which can provide relevant information for future studies.

CD8+ T cell impairment drives severe human metapneumovirus respiratory disease in aged mice.

Abstract Objective Human metapneumovirus (HMPV) is a leading cause of severe respiratory disease in children &amp;lt;5, adults &amp;gt;65, and the immunocompromised. CD8+ T lymphocytes are the primary cell responsible for clearing HMPV infection in the lung. We tested the hypothesis that CD8+ T cell impairment in the aged host drives severe respiratory infection and delayed viral clearance. Methods B6 aged 72–73wk mice and young 6–7wk mice were infected with 2×106 PFU HMPV. Seven days post infection, mice were euthanized and lung, bronchoalveolar lavage (BAL), spleen, and draining lymph nodes were collected for flow cytometry. Lung and BAL supernatant was collected for viral titer, histology, and cytokine measurement. Results Aged mice lost more weight, had delayed viral clearance, and increased clinical scores and lung histopathology. Aged mice produced fewer HMPV-tetramer+ cells in lung and BAL. Aged CD8+ T lymphocytes in BAL co-expressed inhibitory receptors PD-1, TIM-3, LAG-3, and 2B4. Aged CD8+ T lymphocytes also upregulated senescence markers, T-bet and EOMES. Upon ex vivo peptide stimulation, aged CD8+ T cells produced significantly less granzyme B compared to young CD8+ T cells. Adoptive transfer of young T cells into aged mice rescued this severe disease phenotype. Young CD8+ cells in the aged host produced more tetramer+ cells and granzyme B and had less senescent marker expression. Conclusion Aged mice exhibit more severe respiratory disease when infected with HMPV. Aged CD8+ T cells appear to be impaired and upregulate senescence markers that contribute to severe HMPV infection in the aged host. Supported by: NIH/NIAID 5R01 AI085062-09, John V. Williams, MD NIH T32GM008208 Medical Scientist Training Program, University of Pittsburgh School of Medicine

Clinical characteristics and prognostic factors of fibrotic nonspecific interstitial pneumonia.

Aim:Several studies have reported favorable outcomes of nonspecific interstitial pneumonia (NSIP); however, its prognosis and prognostic factors remain unclear. This study aimed to determine the outcomes of fibrotic NSIP and the prognostic factors for progression, relapse, and survival.Methods:In this retrospective study, we reviewed the clinical data of 204 patients diagnosed with fibrotic NSIP by surgical lung biopsy at Samsung Medical Center. The factors associated with survival and disease progression or relapse were determined using Cox proportional hazard analysis.Results:The median age of patients was 54 years and 67 (33%) patients were male. Also, 47 patients (23%) were current or ex-smokers. In all, 141 (69%) patients were diagnosed with idiopathic NSIP, while 63 (31%) patients were associated with connective tissue diseases. Progression or relapse was observed in 100 (49%) patients. The 5-year and 10-year survival rates were 94.6% and 90.4%, respectively. The factors associated with disease progression and relapse were diffusing capacity for carbon monoxide (DLco) <60% [adjusted hazard ratio (HR), 1.739; 95% confidence interval (CI), 1.036–2.921; p = 0.036], bronchoalveolar lavage (BAL) lymphocyte >15% (adjusted HR, 0.592; 95% CI, 0.352–0.994; p = 0.047), and treatment with corticosteroid and azathioprine (adjusted HR, 0.556; 95% CI, 0.311–0.955; p = 0.048). Disease progression or relapse was associated with mortality (adjusted HR, 7.135; 95% CI, 1.499–33.971; p = 0.014).Conclusion:Preserved lung function, BAL lymphocytosis, and treatment with corticosteroids and azathioprine were associated with lower risks of disease progression and relapse, which were risk factors for mortality.

Additive effects of orchiectomy and intermittent hypoxia on lung mechanics and inflammation in C57BL/6J male mice.

What is the central question of this study? Does endogenous testosterone modulate the consequences of intermittent hypoxia (IH) in the lungs of male mice? What is the main finding and its importance? Orchiectomized mice exposed to IH develop a pattern that is similar to emphysema or obstructive lung disease with elevated lung volumes, low pulmonary elastance during a methacholine challenge test and high counts of lymphocytes in bronchoalveolar lavages. Since low testosterone levels and other respiratory diseases are common in sleep apnoea, there is a clear clinical relevance to these results. We tested the hypothesis that low testosterone levels modulate the pulmonary responses to intermittent hypoxia (IH; used as a model of sleep apnoea (SA)) in male mice. We used intact (SHAM) or orchiectomized (ORX) mice exposed to IH for 14days (12h/day, 10cycles/h, 6% oxygen) or to normoxia (Nx). We first measured ventilation and metabolic rates in freely behaving mice (whole-body plethysmography) and then respiratory mechanics in tracheotomized mice (flexiVent). We assessed the respiratory system resistance and elastance (Ers ), Newtonian resistance (resistance of the large airways), tissue damping and tissue elastance (H) under baseline conditions and during a methacholine challenge test. We also measured the quasi-static compliance and inspiratory capacity with partial pressure-volume loops. Finally, inflammatory cells were counted in the broncho-alveolar lavage (BAL) and we measured lung volume by water displacement. ORX-IH mice had higher tidal volume, inspiratory capacity and lung volume compared to the other groups, but showed signs of low efficiency of O2 exchange rate relative to minute ventilation. During the methacholine challenge, orchiectomy decreased the values of most mechanical parameters and IH reduced Ers and H leading to very low values in ORX-IH mice. Finally, the total number of cells and the number of lymphocytes in BAL were both increased by IH in ORX mice. Since reduced lung elasticity, low O2 extraction, increased lung volumes and inflammation are signs of emphysematous lung disease, we conclude that testosterone might prevent lung emphysema during IH exposures.

Antigen avoidance and outcome of nonfibrotic and fibrotic hypersensitivity pneumonitis.

BackgroundHypersensitivity pneumonitis (HP) is classified into nonfibrotic and fibrotic phenotypes. Patients with nonfibrotic HP often experience recurrence and develop fibrosis, whereas those with fibrotic HP have a poor prognosis. Although antigen avoidance has long been the first line of treatment for HP, its impact on prognosis has been poorly reported.MethodsMedical records of 121 patients with HP diagnosed by new diagnostic criteria of American Thoracic Society/Japanese Respiratory Society/Asociación Latinoamericana del Tórax (ATS/JRS/ALAT) guidelines and treated at our institution in Saitama, Japan, were retrospectively analysed. HP was classified into nonfibrotic and fibrotic phenotypes and six HP subtypes: summer-type, bird-related, home-related and occupational HP, humidifier lung, and hot tub lung. Achievement of reduced exposure to inciting agents was divided into complete antigen avoidance (CAA) and incomplete antigen avoidance (IAA) by HP subtype.ResultsOf the 74 patients with nonfibrotic HP, 30 achieved CAA and experienced no recurrence or development of fibrosis. In the remaining 44 patients with IAA, 24 (54.5%) experienced recurrence and/or development of fibrosis. The all-cause 5-year mortality rate in the 47 patients with fibrotic HP was 47.8%. Negative prognostic factors of HP-related mortality in these patients were <50% lymphocytes in bronchoalveolar lavage (BAL) and honeycombing. Multivariate analysis showed a tendency for IAA to be related to poorer survival (hazard ratio 3.452, 95% CI 0.964–12.359, p=0.057).ConclusionsIn the patients with nonfibrotic HP, CAA resulted in no recurrence or development of fibrosis and longer survival. In the patients with fibrotic HP, <50% lymphocytes in BAL and honeycombing were negative prognostic factors for mortality.

Inhibitory effects of somatostatin analogue in bleomycin-induced pulmonary fibrosis

Background and objectives Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease. An increased expression of somatostatin receptor subtype 2 in patients with IPF was identified and lung fibroblasts expressed somatostatin receptors in vitro. In addition, somatostatin analogue inhibits the expression of transforming growth factor-β, insulin-like growth factor (IGF) −1, platelet-derived growth factor, and basic fibroblast growth factor. Therefore, we examined the effects of somatostatin analogue on bleomycin-induced pulmonary fibrosis in mice. In a similar model, it has been reported that administration of high-dose somatostatin analogs suppressed acute inflammation and subsequent pulmonary fibrosis. However, it was clarified that the same effect can be obtained even at the dose used in clinical practice. Methods C57BL/6 mice received a single tracheal instillation of bleomycin. After randomly allocated, mice were treated with subcutaneous injection of either normal saline or somatostatin analogue. Results Somatostatin analogue reduced the number of neutrophils and lymphocytes in bronchoalveolar lavage (BAL) and IGF-1 level in serum and BAL fluid and attenuated weight loss. The hydroxyproline content of the lung homogenates in somatostatin analogue treatment group was significantly lower than in that of normal saline treatment group. Conclusions These results suggest that somatostatin analogue may attenuate pulmonary fibrosis after bleomycin treatment at the dose used in clinical practice.

Comprehensive evaluation of bronchoalveolar lavage from patients with severe COVID-19 and correlation with clinical outcomes

Information on bronchoalveolar lavage (BAL) in patients with COVID-19 is limited, and clinical correlation has not been reported. This study investigated the key features of BAL fluids from COVID-19 patients and assessed their clinical significance. A total of 320 BAL samples from 83 COVID-19 patients and 70 non-COVID-19 patients (27 patients with other respiratory viral infections) were evaluated, including cell count/differential, morphology, flow cytometric immunophenotyping, and immunohistochemistry. The findings were correlated with clinical outcomes. Compared to non-COVID-19 patients, BAL from COVID-19 patients was characterized by significant lymphocytosis (p < 0.001), in contrast to peripheral blood lymphopenia commonly observed in COVID-19 patients and the presence of atypical lymphocytes with plasmacytoid/plasmablastic features (p < 0.001). Flow cytometry and immunohistochemistry demonstrated that BAL lymphocytes, including plasmacytoid and plasmablastic cells, were composed predominantly of T cells with a mixture of CD4+ and CD8+ cells. Both populations had increased expression of T-cell activation markers, suggesting important roles of helper and cytotoxic T-cells in the immune response to SARS-CoV-2 infection in the lung. More importantly, BAL lymphocytosis was significantly associated with longer hospital stay (p < 0.05) and longer requirement for mechanical ventilation (p < 0.05), whereas the median atypical (activated) lymphocyte count was associated with shorter hospital stay (p < 0.05), shorter time on mechanical ventilation (p < 0.05) and improved survival. Our results indicate that BAL cellular analysis and morphologic findings provide additional important information for diagnostic and prognostic work-up, and potential new therapeutic strategies for patients with severe COVID-19.

MANAGEMENT OF AIRWAY REMODELING IN A MOUSE MODEL OF ALLERGIC AIRWAY INFLAMMATION ASSOCIATING HUMAN DENTAL PULP STEM CELLS AND MODERATE AEROBIC TRAINING

The aim of this study was to evaluate the therapeutic potential of human dental pulp stem cells, associated or not with a protocol of moderate aerobic treadmill training, in an ovalbumin(OVA) protocol of allergic airway inflammation in Balb/C mice. This study was approved by Ethics Committee (number: 4.429.378) and Ethics Committee on Animal Use (number: 01242). 60 male Balb/C mice were divided in 2 groups: Aerobic Training (AT, n=30); and without aerobic training (NT, n=30) those divided in 3: Control (C-AT and C-NT, n=10), Airway Inflammation (AI-AT and AI-NT, n=10); and Treatment (Tx-AT and Tx-NT, n=10). AI and Tx groups were submitted to OVA protocol by intraperitoneal injections of OVA at days 0,2,4,7,9 and 10. Then these animals received by IT injections OVA at days 15,18 and 21. C groups only sterile saline. At day 22, animals received hDPSCs by IT administration, while C and AS groups received sterile saline. At day 28 to 30 AT groups were adapted to treadmill (15 min, 0.2 km/h). On the next day (31) and at day 63, a fatigue test performed with warm-up (5 min, 0.2 km/h) followed by an increase in treadmill speed (0.1 km/h every 2.5 min) until exhaustion. Then the animals were trained for 3 days/week for 4 weeks, with 15 minutes warm-up (25% inclination 0.2 km/h) and for 45 minutes, at 50% max speed achieved in fatigue test. At day 64, we performed bronchoalveolar lavage (BAL) to count total nuclear cells. The animals were euthanized using anesthetic overdose, and lungs were collected for histopathological analysis (HE, MT and PAS staining). Data were analyzed using Kruskal-Wallis test followed by Dunn's test for multiple comparisons, considering significant P<0.05. At this moment, animals from Tx groups were not performed yet. Animals from both AS groups showed an increase on total cell count on BAL fluid, eosinophil and lymphocytes counts as well, and for macrophage cells counts were decreased when compared to both C groups. VO2 levels were overall higher in the final fatigue test, compared with the initial test, as well for VCO2 levels for all groups with C group showing higher levels of both parameters compared with AS groups. Maximum speed increased at the final fatigue test for all subjects which indicates improvement on physical conditioning. Even without the Tx groups, moderate aerobic training was not harmful and improved physical conditioning in animals with asthma, and did not worsened the disease in those animals.

Computed Tomography Findings as Determinants of Local and Systemic Inflammation Biomarkers in Interstitial Lung Diseases: A Retrospective Registry-Based Descriptive Study

PurposeTo evaluate the association of peripheral blood (PBL) and broncho-alveolar lavage (BAL) biomarkers with inflammatory versus fibrotic high-resolution computed tomography (HRCT) findings in interstitial lung disease (ILD) patients.MethodsHRCT findings of 127 consecutive ILD-board patients were semi-quantitatively evaluated: reticulation/honeycombing (RET), traction bronchiectasis (TBR) and emphysema (EMP) were classified as non-inflammatory/fibrotic; consolidations (CON), ground glass opacities (GGO), parenchymal nodules (NDL) and mosaic attenuation (MOS) as active inflammatory. Each HRCT finding was assessed in six distinct lung regions, resulting scores were graded as minimal (0–1 regions involved), medium (2–4) or extensive (5–6). Associations of routinely assessed PBL/BAL biomarkers with these HRCT scores were evaluated using Spearman correlation coefficients and graphical presentation; significance was tested by applying Kruskal–Wallis tests.ResultsBlood neutrophil, lymphocyte and eosinophil fraction, neutrophil to lymphocyte ratio (NLR) and BAL lymphocyte fraction consistently showed opposite correlations with inflammatory versus non-inflammatory/fibrotic HRCT finding scores. Blood lymphocyte fraction significantly differed by graded GGO (p = 0.032) and CON (p = 0.027) extent, eosinophil fraction by TBR (p = 0.006) and NLR by CON (p = 0.009). C-reactive protein was significantly related to GGO (p = 0.023) and CON (p = 0.004), BAL lymphocyte fraction to GGO (p = 0.017) extent.ConclusionBlood lymphocyte and eosinophil fraction, NLR, CRP and BAL lymphocyte fraction may aid to differentiate inflammatory from non-inflammatory/fibrotic ILD patterns.Trial registrationThis evaluation was based on data from the ILD registry of Kepler University Hospital Linz, as approved by the ethics committee of the Federal State of Upper-Austria (EK Number. I-26-17).

Hypersensitivity pneumonitis – acceptance of the diagnosis

Abstract Hypersensitivity pneumonia (HP) is an inflammatory lung syndrome due to repetitive exposure to small organic particles. The etiological agents of the disease are diverse and can be represented by fungi, bacteria, small molecule chemical compounds, as well as proteins from animals or insects. We present the case of a 25-year-old male, who was admitted to our Pulmonology Clinic for the following symptoms: morning cough with mucosal sputum for about 2 years, moderate exertional dyspnea (mMRC 3) started for 2 months, weight loss of about 6 kg in the past 5 months and fatigue. Clinical examination revealed bilateral mid- basal crackling rales. Chest high-resolution computed tomography (HRCT) (multiple micronodular opacities, diffuse contoured, bilaterally disseminated), lung function tests (restrictive ventilatory dysfunction with decreased carbon monoxide diffusing capacity), bronchoalveolar lavage (BAL) (lymphocyte alveolitis) and serological tests (positive antibodies for pigeon heathers and dejections) pointed to the diagnosis of hypersensitivity pneumonitis. It was recommended to avoid exposure to pigeons, as the patient was a pigeon breeder. Prednisone treatment was initiated. The general condition of the patient 1 month after diagnosis had improved, the evolution being good under treatment.

Bronchoalveolar Lavage Lymphocytes in the Diagnosis of Hypersensitivity Pneumonitis among Patients with Interstitial Lung Disease

Rationale: Hypersensitivity pneumonitis (HP) is an interstitial lung disease (ILD) characterized by inflammation and/or fibrosis in response to an inhalational exposure.Objectives: To determine the value of bronchoalveolar lavage (BAL) fluid lymphocyte cellular analysis in the detection of HP among patients with newly detected ILD.Methods: This systematic review was undertaken in the context of development of an American Thoracic Society, Japanese Respiratory Society, and Asociación Latinoamericana del Tórax clinical practice guideline. The clinical question was, "should patients with newly detected ILD undergo BAL fluid lymphocyte analysis to diagnose HP?" MEDLINE, EMBASE, and the gray literature were searched through October 2019. Studies that reported the percentage of BAL fluid lymphocytes for various ILDs were selected for inclusion. Meta-analyses compared the mean percentage of BAL fluid lymphocytes among patients with HP with that among patients with idiopathic pulmonary fibrosis (IPF) or sarcoidosis. The sensitivity and specificity by which various percentages of BAL fluid lymphocytes distinguish HP from IPF and sarcoidosis were also evaluated.Results: Eighty-four articles were selected. No randomized trials or observational studies were identified that compared BAL fluid lymphocyte analysis with no BAL fluid lymphocyte analysis in patients with ILD. Included studies were case series describing BAL fluid cell differentials in patients with various ILDs. The percentage of BAL fluid lymphocytes was significantly higher in both fibrotic and nonfibrotic HP compared with IPF. Similarly, the percentage of BAL fluid lymphocytes was significantly higher in both fibrotic and nonfibrotic HP compared with sarcoidosis. A threshold of 20% BAL fluid lymphocytes distinguished fibrotic HP from IPF with a sensitivity and specificity of 69% and 61%, respectively, and nonfibrotic HP from IPF with a sensitivity and specificity of 95% and 61%, respectively. It distinguished fibrotic HP from sarcoidosis with a sensitivity and specificity of 69% and 26%, respectively, and nonfibrotic HP from sarcoidosis with a sensitivity and specificity of 95% and 26%, respectively.Conclusions: The percentage of BAL fluid lymphocytes is higher in HP than IPF or sarcoidosis. However, a threshold that distinguishes HP from IPF or sarcoidosis with both high sensitivity and high specificity was not identified.

Clinical significance of serum anti-granulocyte\u2013macrophage colony-stimulating factor autoantibodies in patients with sarcoidosis and hypersensitivity pneumonitis

BackgroundAnti-granulocyte–macrophage colony-stimulating factor autoantibody (GMAb) has been recognized as a diagnostic biomarker for autoimmune pulmonary alveolar proteinosis (aPAP). The aims of this study were to know the incidence of increased level of serum GMAb in granulomatous lung diseases (sarcoidosis and hypersensitivity pneumonitis [HP]) and to clarify the role of GMAb. Consecutive individuals diagnosed with sarcoidosis (n = 92) and HP (n = 45) at National Hospital Organization Kinki-Chuo Chest Medical Center were retrospectively analyzed. We measured serum GMAb levels at the diagnosis. Cut-off values of GMAb discriminating aPAP (n = 110) from healthy controls (n = 31) were determined by receiver operating characteristic (ROC) curve analysis. We compared the clinical features of sarcoidosis and HP patients with GMAb levels above the cut-off value (“Elevated-GMAb”) with those of patients whose GMAb levels below the cut-off value (“Low-GMAb”). Radiological and pathological findings in elevated-GMAb patients were re-evaluated to elucidate the role of GMAb in granulomatous lung diseases.ResultsAnalysis of ROC indicated a sensitivity and specificity of 100% at GMAb level of 3.33 μg/mL for discriminating aPAP from healthy controls (area under curve = 1.000, p < 0.0001). The percentages of elevated-GMAb sarcoidosis and HP patients were 5.4% (n = 5) and 11.1% (n = 5), respectively. The number of comorbid sarcoidosis and HP patients with aPAP was two and one, respectively. Elevated-GMAb sarcoidosis patients presented with significantly higher serum levels of Krebs von den Lungen (KL)-6, surfactant protein-D (SP-D), lactate dehydrogenase, and the requirement of systemic corticosteroid therapy. Elevated-GMAb HP patients demonstrated older age, higher serum KL-6, SP-D, carcinoembryonic antigen, and cytokeratin fragment 21-1 levels, and a higher percentage of lymphocytes in bronchoalveolar lavage than low-GMAb patients. A subset of patients presented with radiological and pathological findings characteristic of aPAP.ConclusionsWe demonstrated the percentage of elevated-GMAb sarcoidosis and HP patients who presented with several features suggestive of aPAP. Elevated-GMAb sarcoidosis and HP patients without definitive aPAP diagnosis may have subclinical or early-stage aPAP and may not necessarily indicate false positives. Upon diagnosis of sarcoidosis or HP, measurement of GMAb may be useful in detecting possible comorbidity of subclinical or early-onset aPAP.