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Nicole MD van der Burg, Carl Ekelund, Leif H Bjermer, David Aronsson, Jaro Ankerst, Ellen Tufvesson Department of Clinical Sciences Lund, Respiratory Medicine, Allergology and Palliative Medicine, Lund University, Lund, SwedenCorrespondence: Ellen Tufvesson, Department of Clinical Sciences Lund, Respiratory Medicine, Allergology and Palliative Medicine, Lund University, BMC, D12, Lund, 221 84, Sweden, Email Ellen.Tufvesson@med.lu.seBackground: Bronchodilator responsiveness (BDR) in asthma involves both the central and peripheral airways but is primarily relieved with beta-2-agonists and evaluated by spirometry. To date, antimuscarinics can be added as a reliever medication in more severe asthma. We hypothesize that combining both short-acting beta-2 agonist (SABA) and short-acting muscarinic antagonist (SAMA) could also improve the responsiveness in mild-moderate asthma. Therefore, we aimed to compare the direct effects of inhaling SABA alone, SAMA alone or combining both SABA and SAMA on the central and peripheral airways in asthma.Methods: Twenty-three patients with mild-moderate BDR in asthma performed dynamic spirometry and impulse oscillometry before (baseline) and multiple timepoints within an hour after inhalation of SABA (salbutamol), SAMA (ipratropium bromide), or both SABA and SAMA at three different visits.Results: The use of SAMA alone did not show any improvement compared to the use of SABA alone. Inhalation of SABA+SAMA, however, averaged either similar or better BDR than SABA alone in FEV1, MMEF, FVC, R5, R20 and R5-R20. Inhaling SABA+SAMA reached a stable BDR in more patients within 0– 10 minutes and also reached the FEV1 (Δ%)> 12% faster (3.5 minutes) than inhaling SABA alone (5.1 minutes). Inhaling SABA+SAMA was significantly better than SAMA alone in FEV1 (p = 0.015), MMEF (p = 0.0059) and R20 (p = 0.0049). Using these three variables highlighted a subgroup (30%, including more males) of patients that were more responsive to inhaling SABA+SAMA than SABA alone.Conclusion: Overall, combining SAMA with SABA was faster and more consistent at increasing the lung function than SABA alone or SAMA alone, and the additive effect was best captured by incorporating peripheral-related variables. Therefore, SAMA should be considered as an add-on reliever for mild-moderate patients with BDR in asthma.Keywords: BDR, mild-moderate asthma, central airway, peripheral airways, SABA, SAMA

Martin Garcia-Ryde,1 Nicole MD van der Burg,1 Carin E Larsson,1 Anna-Karin Larsson-Callerfelt,2 Gunilla Westergren-Thorsson,2 Leif Bjermer,1 Ellen Tufvesson1 1Department of Clinical Sciences Lund, Respiratory Medicine, Allergology and Palliative Medicine, Lund University, Lund, Sweden; 2Department of Experimental Medical Science, Lung Biology, Lund University, Lund, SwedenCorrespondence: Ellen Tufvesson, Department of Clinical Sciences Lund, Respiratory Medicine, Allergology and Palliative Medicine, Lund University, BMC, D12, 221 84, Lund, Sweden, Email Ellen.Tufvesson@med.lu.seBackground and aim: Cigarette smoking is the most common cause of chronic obstructive pulmonary disease (COPD) but more mechanistic studies are needed. Cigarette smoke extract (CSE) can elicit a strong response in many COPD-related cell types, but no studies have been performed in lung fibroblasts. Therefore, we aimed to investigate the effect of CSE on gene expression in lung fibroblasts from healthy and COPD subjects.Patients and methods: Primary lung fibroblasts, derived from six healthy and six COPD subjects (all current or ex-smokers), were either unstimulated (baseline) or stimulated with 30% CSE for 4 h prior to RNA isolation. The mRNA expression levels were measured using the NanoString nCounter Human Fibrosis V2 panel (760 genes). Pathway enrichment was assessed for unique gene ontology terms of healthy and COPD.Results: At baseline, a difference in the expression of 17 genes was found in healthy and COPD subjects. Differential expression of genes after CSE stimulation resulted in significantly less changes in COPD lung fibroblasts (70 genes) than in healthy (207 genes), with 51 genes changed in both. COPD maintained low NOTCH signaling throughout and upregulated JUN > 80%, indicating an increase in apoptosis. Healthy downregulated the Mitogen-activated protein kinase (MAPK) signaling cascade, including a ≥ 50% reduction in FGF2, CRK, TGFBR1 and MEF2A. Healthy also downregulated KAT6A and genes related to cell proliferation, all together indicating possible cell senescence signaling.Conclusion: Overall, COPD lung fibroblasts responded to CSE stimulation with a very different and deficient expression profile compared to healthy. Highlighting that stimulated healthy cells are not an appropriate substitute for COPD cells which is important when investigating the mechanisms of COPD.Plain Language Summary: We investigated, for the first time, the effect of cigarette smoke extract (CSE) on gene expression in lung fibroblasts from healthy subjects and subjects with COPD. We found that fibroblasts from subjects with COPD respond very differently than from healthy subjects by changing fewer gene expressions after CSE. Pathway enrichment suggested that fibroblasts from subjects with COPD respond to CSE by affecting genes involved in apoptosis pathways, while fibroblasts from healthy subjects respond by affecting genes involved in cell senescence. This study highlights the importance of assessing stimulated COPD cells in mechanistic studies.Keywords: COPD, lung fibroblast, gene expression, cigarette smoke extract, GO term, signaling pathways

Ellen Tufvesson,1 Finn Radner,1 Georgia Papapostolou,1 Linnea Jarenbäck,1 Saga Jönsson,1 Ulf Nihlén,1 Jaro Ankerst,1 Alf Tunsäter,1 Stefan Peterson,2 Leif Bjermer,1 Göran Eriksson1 1Department of Clinical Sciences Lund, Respiratory Medicine and Allergology, Lund University, Skane University Hospital, Lund, Sweden; 2Regional Cancer Centre South, Region Skane, Lund, SwedenCorrespondence: Ellen TufvessonDepartment of Clinical Sciences Lund, Respiratory Medicine and Allergology, Lund University, Skane University Hospital, Lund 22185, SwedenTel +46 73 640 19 16Email ellen.tufvesson@med.lu.sePurpose: For exercise testing of COPD patients, a standard endurance test (ET) with constant workload is recommended. The test suffers from large inter-individual variability and need for large sample sizes in order to evaluate treatment effects.Methods: A new protocol for ET in COPD was designed. In contrast to the standard ET, the new ET involved an increasing workload in order to reduce the standard deviation of endurance time. Two new ETs were compared with the standard ET. In Study A, the new ET started at 75% of the patient’s maximum workload (WMAX) and increased stepwise with 3%/2 min until exhaustion. Study B started at 70% of WMAX and increased linearly with 1%/min.Results: In Study A, that included 15 patients, the standard deviation and range for endurance time and work capacity were narrower for the new versus the standard ET. However, the higher mean workload at end and the low mean work capacity relative to the standard ET indicated that the stepwise increase was too aggressive. In Study B, that included 18 patients, with a modified protocol, the averages for endurance time, workload at end and work capacity were similar for new and standard ET, while the standard deviations and ranges for endurance time and work capacity were kept more narrow in the new ET. The variances for endurance time were not equal between the standard ET and the two new ETs (p< 0.05 for both according to Levene’s test).Conclusion: The new ET reduced the number of patients with extreme endurance times (short and long) compared to the standard test. The new test showed a significant lower variance for endurance time, which potentially can lead to fewer patients needed in comparative studies. The overall best results were observed with a low linear increase during endurance.Keywords: COPD, exercise testing, constant endurance test, endurance time

Ellen Tufvesson, Henning Stenberg, Jaro Ankerst, Leif Bjermer Respiratory Medicine and Allergology, Department of Clinical Sciences Lund, Lund University, Lund, SwedenCorrespondence: Ellen Tufvesson Respiratory Medicine and Allergology, Department of Clinical Sciences LundLund University, Lund 221 84, SwedenTel +46 736 401916Email Ellen.Tufvesson@med.lu.seIntroduction: Exercise-induced bronchoconstriction is due to osmotic stimulus of the airway epithelium and leads to a cascade of biomarker release from several inflammatory cells. Several type 2 (T2) mediators have been linked to exercise-induced bronchoconstriction, but the T2 response per se has not been described during exercise. The aim of this study was therefore to investigate T2 biomarkers in plasma and urine from subjects with asthma and healthy controls before and after an exercise challenge.Methods: Twenty-two subjects with mild asthma and 18 healthy controls performed an exercise challenge test on a treadmill, and fractional exhaled NO (FeNO) was measured at baseline. Blood and urine samples were collected repeatedly during 60 min after the test and Interleukin-13 (IL-13), thymus and activation-related chemokine (TARC), periostin and leukotrienes were measured.Results: Asthmatics and controls showed similar levels of IL-13, TARC, periostin and Cys-LT in plasma at baseline, and there were no differences in baseline levels between subjects with a negative and positive exercise challenge. After exercise, there was an overall increase in interleukin-13 (IL-13) in plasma in all subjects (p< 0.001), with a peak at 10 min after the exercise challenge in both the asthmatic and control group. An increase in TARC in plasma was also seen (p< 0.001), but only in the control subjects. In contrast, Cys-LT in plasma showed an overall decrease in all subjects (p< 0.001), while periostin in plasma did not change. In conjunction with plasma, the level of IL-13 was increased in urine 30 min after the exercise challenge (p=0.002) and decreased again at 60 min (p=0.004). Similarly, leukotriene E4 (LTE4) was increased in urine samples, with a peak at 60 min and most pronounced in asthmatic subjects (p< 0.001) but was seen also in controls (p=0.008).Discussion: In conclusion, circulating levels of IL-13 are increased after exercise to the same extent in asthmatics and healthy control subjects, which indicates a physiological rather than a pathophysiological response. Also, the levels of TARC and leukotrienes were affected after exercise.Keywords: asthma, biomarkers, exercise, inflammation

Linnea Jarenbäck,1 Ellen Tufvesson,1 Jaro Ankerst,1 Leif Bjermer,1 Björn Jonson2 1Respiratory Medicine and Allergology, Department of Clinical Sciences, Skåne University Hospital, Lund University, Lund, Sweden; 2Clinical Physiology, Department of Clinical Sciences, Skåne University Hospital, Lund University, Lund, Sweden Background: Spirometry, the main tool for diagnosis and follow-up of COPD, incompletely describes the disease. Based on volumetric capnography (VCap), an index was developed for the diagnosis and grading of COPD, aimed as a complement or alternative to spirometry.Methods: Nine non-smokers, 10 smokers/former smokers without COPD and 54 smokers/former smokers with COPD were included in the study. Multiple breath washout of N2 and VCap were studied with Exhalyzer D during tidal breathing. VCap was based on signals for flow rate and CO2 and was recorded during one breath preceding N2 washout. Efficiency Index (EFFi) is the quotient between exhaled CO2 volume and the hypothetical CO2 volume exhaled from a completely homogeneous lung over a volume interval equal to 15% of predicted total lung capacity.Results: EFFi increased with increased Global initiative for chronic Obstructive Lung Disease (GOLD) stage and the majority of subjects in GOLD 2 and all subjects in GOLD 3 and 4 could be diagnosed as having COPD using the lower 95% confidence interval of the healthy group. EFFi also correlated with N2 washout (r=–0.73; p

Tufvesson E, Radner F, Papapostolou G, et al. Int J Chron Obstruct Pulmon Dis. 2020;15:3003–3012. Page 3004, left column, line 2 from the bottom, the sentence “The study also included a new WMAX test where the participants started biking with a 3-min warm-up at 40% of their predicted WMAX, followed by an instant increase to 70% and then a linear increase in workload to achieve the predicted WMAX within 8 min” should read “The study also included a new WMAX test where the participants started biking with a 3-min warm-up at 40% of their predicted WMAX, followed by a linear increase in workload to achieve the predicted WMAX within 8 min”. Page 3005, left column, line 4 from the bottom, the sentence “The new maximum test included an initial phase of pedalling for 3 min at 40% (Study A) or 30% (Study B) of the predicted WMAX, followed by an instant increase to 75% (Study A) or 70% (Study B), followed by a stepwise (Study A) or linear (Study B) increase in workload until exhaustion” should read “The new maximum test included an initial phase of pedalling for 3 min at 40% (Study A) or 30% (Study B) of the predicted WMAX, followed by a linear increase in workload to achieve the predicted WMAX within 8 min”. The authors apologize for these errors. Read the original article

Ellen Tufvesson,1 Hanna Markstad,2 Gracijela Bozovic,2 Marie Ekberg,1 Leif Bjermer1 1Respiratory Medicine and Allergology, 2Radiology, Department of Clinical Sciences Lund, Lund University, Skåne University Hospital, Lund, Sweden Abstract: The presence of bacteria in the lower airways in COPD results in inflammation, further airway structural damage, and might lead to repeated exacerbations. We have previously shown that chronic colonization of Haemophilus influenzae during stable disease is related to increased inflammation, and we now aimed to relate previous findings of bacterial colonization and inflammation to the degree of radiological findings of bronchiectasis and emphysema. Thirty-nine patients with COPD were included in their stable state, and a high-resolution computed tomography of the lung was performed. They were followed-up monthly for up to a maximum of 6 months or until exacerbation, and they answered questionnaires, performed spirometry, and induced sputum at every visit. Thirty-five patients had emphysema with an emphysema degree of median 20% (interquartile range 10–50), and five patients had bronchiectasis, of which only four could expectorate sputum. The degree of emphysema correlated with several inflammatory mediators in sputum, such as interleukin-8 concentration, myeloperoxidase activity, and Leukotriene B4 concentration. Ten patients were chronically colonized with H. influenzae (ie, had a positive culture for H. influenzae at all visits). The four sputum patients with bronchiectasis were chronically colonized with H. influenzae and showed higher degree of H. influenzae growth compared to patients without bronchiectasis. During exacerbation, there was no longer any correlation between emphysema degree and inflammation, but patients with bronchiectasis showed higher sputum purulence score than patients without bronchiectasis. Emphysema and bronchiectasis in COPD patients show different clinical features. The presence of emphysema is more related to inflammation, while bronchiectasis is associated with bacterial colonization. We believe that both emphysema and bronchiectasis are therefore COPD phenotypes of highest impact and need evaluation to prevent further disease progression. Keywords: bronchiectasis, emphysema, COPD, chronic colonization, inflammation, pathogenic bacteria

Kerstin Romberg,1,2 Ellen Tufvesson,1 Leif Bjermer1 1Respiratory Medicine and Allergology, Department of Clinical Sciences, Lund, Lund University, Lund, 2Health Care Center, Näsets Läkargrupp, Höllviken, Sweden Background: Strenuous physical activity at an elite level is associated with an increased risk for asthma and, in some sports, also prevalence of allergies. The aim of this study was to investigate the prevalence of asthma and allergy among elite swimmers and tennis players and compare airway hyperreactivity to mannitol and exercise.Materials and methods: One hundred and one adolescent swimmers and 86 tennis players answered a questionnaire about respiratory symptoms and allergy and performed mannitol challenge and sport-specific exercise challenge. Atopy was assessed and fractional exhaled nitric oxide was measured. Mannitol positivity was defined as drop in FEV1 ≥15% (ordinary criteria) and/or β2-reversibility (≥15%) after provocation (extended criteria). A positive exercise test was defined as a drop in FEV1 ≥10% (ordinary criteria) and/or β2-reversibility (≥15%) after provocation (extended criteria). Club cell protein (CC16) was measured in urine before and after the challenges.Results: Asthma symptoms were common in both groups. More swimmers had exercise-induced symptoms (77% versus 50%) and current asthma symptoms (56% versus 38%), compared to the tennis players. More swimmers also had a positive mannitol challenge test both using ordinary (26% versus 6%) and extended criteria (43% versus 17%), while the number of positive exercise tests did not differ. After exercise (but not mannitol) challenge, CC16 level was increased in both groups, but to a higher extent in tennis players. There were no differences in atopy, rhinitis or fractional exhaled nitric oxide.Conclusion: We found a high prevalence of asthma among elite swimmers and tennis players and a higher frequency of current asthma and positive mannitol challenge tests among the swimmers. This indicates an unfavorable exercise environment. Keywords: asthma, sport, swimming, tennis, mannitol, exercise, CC16 bronchial hyperreactivity, bronchial hyperreactivity test

Linnea Jarenbäck,1 Göran Eriksson,1 Stefan Peterson,2 Jaro Ankerst,1 Leif Bjermer,1 Ellen Tufvesson1 1Respiratory Medicine and Allergology, Department of Clinical Sciences Lund, Lund University, 2Regional Cancer Center South, Skåne University Hospital, Lund, Sweden Background: COPD is defined as partly irreversible airflow obstruction. The response pattern of bronchodilators has not been followed in advanced lung function parameters. Purpose: The aim of this study was to investigate bronchodilator response pattern in advanced lung function parameters in a continuous fashion along forced expiratory volume in 1 second (FEV1) percent predicted (%p) in COPD patients and controls. Patients and methods: Eighty-one smokers/ex-smokers (41 controls and 40 COPD) performed spirometry, body plethysmography, impulse oscillometry and single-breath helium dilution carbon monoxide diffusion at baseline, after salbutamol inhalation and then after an additional inhalation of ipratropium. Results: Most pulmonary function parameters showed a linear increase in response to decreased FEV1%p. The subjects were divided into groups of FEV1%p 65, and the findings from continuous analysis were verified. The exceptions to this linear response were inspiratory capacity (IC), forced vital capacity (FVC), FEV1/FVC and expiratory resistance (Rex), which showed a segmented response relationship to FEV1%p. IC and FVC, with break points (BP) of 57 and 58 FEV1%p respectively, showed no response above, but an incresed slope below the BP. In addition, in patients with FEV1%p 65, response of FEV1%p did not correlate to response of volume parameters. Conclusion: Response of several advanced lung function parameters differs depending on patients’ baseline FEV1%p, and specifically response of volume parameters is most pronounced in COPD patients with FEV1%p

Göran Eriksson,1,* Linnea Jarenbäck,1,* Stefan Peterson,2 Jaro Ankerst,1 Leif Bjermer,1 Ellen Tufvesson11Respiratory Medicine and Allergology, Department of Clinical Sciences, Lund University, 2Regional Cancer Center South, Skåne University Hospital, Lund, Sweden*These authors contributed equally to this workPurpose: COPD is a progressive disease, which can take different routes, leading to great heterogeneity. The aim of the post-hoc analysis reported here was to perform continuous analyses of advanced lung function measurements, using linear and nonlinear regressions.Patients and methods: Fifty-one COPD patients with mild to very severe disease (Global Initiative for Chronic Obstructive Lung Disease [GOLD] Stages I–IV) and 41 healthy smokers were investigated post-bronchodilation by flow-volume spirometry, body plethysmography, diffusion capacity testing, and impulse oscillometry. The relationship between COPD severity, based on forced expiratory volume in 1 second (FEV1), and different lung function parameters was analyzed by flexible nonparametric method, linear regression, and segmented linear regression with break-points.Results: Most lung function parameters were nonlinear in relation to spirometric severity. Parameters related to volume (residual volume, functional residual capacity, total lung capacity, diffusion capacity [diffusion capacity of the lung for carbon monoxide], diffusion capacity of the lung for carbon monoxide/alveolar volume) and reactance (reactance area and reactance at 5Hz) were segmented with break-points at 60%–70% of FEV1. FEV1/forced vital capacity (FVC) and resonance frequency had break-points around 80% of FEV1, while many resistance parameters had break-points below 40%. The slopes in percent predicted differed; resistance at 5 Hz minus resistance at 20 Hz had a linear slope change of -5.3 per unit FEV1, while residual volume had no slope change above and -3.3 change per unit FEV1 below its break-point of 61%.Conclusion: Continuous analyses of different lung function parameters over the spirometric COPD severity range gave valuable information additional to categorical analyses. Parameters related to volume, diffusion capacity, and reactance showed break-points around 65% of FEV1, indicating that air trapping starts to dominate in moderate COPD (FEV1 =50%–80%). This may have an impact on the patient’s management plan and selection of patients and/or outcomes in clinical research.Keywords: spirometry, severity, body plethysmography, single-breath carbon-monoxide diffusion test, impulse oscillometry, break-point

Ellen Tufvesson, Leif Bjermer, Marie Ekberg Respiratory Medicine and Allergology, Department of Clinical Sciences, Lund University, Lund, Sweden Background: Some patients with chronic obstructive pulmonary disease (COPD) show increased airway inflammation and bacterial colonization during stable phase. The aim of this study was to follow COPD patients and investigate chronic colonization with pathogenic bacteria during stable disease phase, and relate these findings to clinical parameters, inflammatory pattern, lung function, and exacerbations. Methods: Forty-three patients with COPD were included while in a stable state and followed up monthly until exacerbation or for a maximum of 6 months. The patients completed the Clinical COPD Questionnaire and Medical Research Council dyspnea scale questionnaires, and exhaled breath condensate was collected, followed by spirometry, impulse oscillometry, and sputum induction. Results: Ten patients were chronically colonized (ie, colonized at all visits) with Haemophilus influenzae during stable phase. These patients had higher sputum levels of leukotriene B4 (P

Jonas Jögi,1 Hanna Markstad,2 Ellen Tufvesson,3 Leif Bjermer,3 Marika Bajc1 1Department of Clinical Physiology and Nuclear Medicine, 2Department of Radiology, 3Department of Respiratory Medicine and Allergology, Skåne University Hospital and Lund University, Lund, Sweden Abstract: Ventilation/perfusion (V/P) single-photon emission computed tomography (SPECT) is recognized as a diagnostic method with potential beyond the diagnosis of pulmonary embolism. V/P SPECT identifies functional impairment in diseases such as heart failure (HF), pneumonia, and chronic obstructive pulmonary disease (COPD). The development of hybrid SPECT/computed tomography (CT) systems, combining functional with morphological imaging through the addition of low-dose CT (LDCT), may be useful in COPD, as these patients are prone to lung cancer and other comorbidities. The aim of this study was to investigate the added value of LDCT among healthy smokers and patients with stable COPD, when examined with V/P SPECT/CT hybrid imaging. Sixty-nine subjects, 55 with COPD (GOLD I–IV) and 14 apparently healthy smokers, were examined with V/P SPECT and LDCT hybrid imaging. Spirometry was used to verify COPD grade. Only one apparently healthy smoker and three COPD patients had a normal or nearly normal V/P SPECT. All other patients showed various degrees of airway obstruction, even when spirometry was normal. The same interpretation was reached on both modalities in 39% of the patients. LDCT made V/P SPECT interpretation more certain in 9% of the patients and, in 52%, LDCT provided additional diagnoses. LDCT better characterized the type of emphysema in 12 patients. In 19 cases, tumor-suspected changes were reported. Three of these 19 patients (ie, 4.3% of all subjects) were in the end confirmed to have lung cancer. The majority of LDCT findings were not regarded as clinically significant. V/P SPECT identified perfusion patterns consistent with decompensated left ventricular HF in 14 COPD patients. In 16 patients (23%), perfusion defects were observed. HF and perfusion defects were not recognized with LDCT. In COPD patients and long-time smokers, hybrid imaging had added value compared to V/P SPECT alone, by identifying patients with lung malignancy and more clearly identifying emphysema. V/P SPECT visualizes comorbidities to COPD not seen with LDCT, such as pulmonary embolism and left ventricular HF. Keywords: lung, lung cancer, V/P SPECT, ventilation/perfusion, single-photon emission computed tomography, computed tomography

Objective: Small airway disease and chronic obstructive pulmonary disease are common in primary Sjögren's syndrome (pSS). However, the underlying inflammatory mechanisms behind pSS-associated airway disease have not been studied in detail. We therefore wanted to study cytokine and leucocyte levels in induced sputum in never-smoking patients with pSS.Method: Induced sputum cytokines and leucocytes were assessed in 20 never-smoking patients with pSS and 19 age- and gender-matched population-based controls. In addition, pulmonary function, disease activity, respiratory symptoms, and inflammatory and serological features of pSS were assessed.Results: B-cell activating factor (BAFF), interleukin-6 (IL-6) and IL-8 were significantly increased in induced sputum in pSS patients compared to population-based controls, while IL-1β, interferon-α, and tumour necrosis factor-α levels and leucocytes were not. The proportion of lymphocytes and BAFF levels in induced sputum correlated significantly in pSS patients. However, cytokine levels in induced sputum were not associated with pulmonary function tests, disease activity, respiratory symptoms, or serological features of pSS.Conclusion: The increase in BAFF, IL-6, and IL-8 in induced sputum suggests a specific ongoing inflammatory disease process in the airways in pSS patients. Its association with pSS-associated airway disease needs to be further examined in future larger studies. [ABSTRACT FROM AUTHOR]

Introduction Increased exhaled nitric oxide ( NO) levels in asthma are suggested to be through inducible NO synthase (i NOS). The aim of this study was to investigate the expression of i NOS in bronchoalveolar lavage ( BAL) cells and tissue from central and peripheral airways and compare it with the exhaled bronchial and alveolar NO levels in patients with asthma vs a control group. Methods Thirty-two patients with asthma (defined as controlled or uncontrolled according to Asthma Control Test score cut-off: 20) and eight healthy controls were included. Exhaled NO was measured, and alveolar concentration and bronchial flux were calculated. i NOS was measured in central and peripheral lung biopsies, as well as BAL cells. Bronchoalveolar lavage macrophages were stimulated in vitro, and i NOS expression and NO production were investigated. Results Expression of i NOS was increased in central airway tissue and the alveolar compartment in uncontrolled as compared to controlled asthmatics and healthy controls. There were no differences, however, in i NOS m RNA levels in total BAL cells in uncontrolled as compared to controlled asthma. Bronchoalveolar lavage cell m RNA levels of i NOS or i NOS expression in central and alveolar tissue did not relate to alveolar NO, nor to bronchial flux of NO. In vitro stimulation with leukotriene D4 increased i NOS m RNA levels and NO production in cultured BAL macrophages. Conclusion The levels of both bronchial and alveolar i NOS are increased in uncontrolled as compared to controlled asthma. However, levels of i NOS in BAL macrophages were not reflected by alveolar NO. Both central and distal i NOS levels may reflect responsiveness to steroid treatment. [ABSTRACT FROM AUTHOR]

Objectives: In systemic sclerosis (SSc)-related interstitial lung disease (ILD), elevated eosinophil counts in bronchoalveolar lavage are associated with a worse outcome. We hypothesized that eosinophils may be activated in the peripheral circulation, thereby increasing their recruitment to affected tissues and contributing to inflammation and fibrosis. The aim of this study was to characterize the blood eosinophils in SSc patients. Method: Expression levels of surface markers CD11b, CD44, CD48, CD54, CD69, CD81, and HLA-DR on CD16(low)CD9(high)-expressing eosinophils were measured by flow cytometry in whole blood from SSc patients (n = 32) and controls (n = 11). Results: Expression levels of CD54, CD69, and HLA-DR were undetectable in all groups. CD44 and CD11b expression levels were similar between groups. CD81 expression was lower in patients compared to controls independent of disease duration (p = 0.001). CD48 expression was increased in patients with a short disease duration (< 2 years) compared to both controls (p = 0.042) and patients with longer disease duration (≥ 2 years; p = 0.027). In patients with short disease duration, increased CD48 expression was associated with alveolar inflammation as measured by an increased concentration of alveolar nitric oxide (r = 0.76, p = 0.003). Conclusions: Blood eosinophils change phenotype during disease evolution in SSc, and CD48 expression may be used as a biomarker for pulmonary inflammation. [ABSTRACT FROM AUTHOR]

Over the past years, eosinophils have become a focus of scientific interest, especially in the context of their recently uncovered functions (e.g. antiviral, anti-inflammatory, regulatory). These versatile cells display both beneficial and detrimental activities under various physiological and pathological conditions. Eosinophils are involved in the pathogenesis of many diseases which can be classified into primary (clonal) and secondary (reactive) disorders and idiopathic (hyper)eosinophilic syndromes. Depending on the biological specimen, the eosinophil count in different body compartments may serve as a biomarker reflecting the underlying pathophysiology and/or activity of distinct diseases and as a therapy-driving (predictive) and monitoring tool. Personalized selection of an appropriate therapeutic strategy directly or indirectly targeting the increased number and/or activity of eosinophils should be based on the understanding of eosinophil homeostasis including their interactions with other immune and non-immune cells within different body compartments. Hence, restoring as well as maintaining homeostasis within an individual's eosinophil pool is a goal of both specific and non-specific eosinophil-targeting therapies. Despite the overall favourable safety profile of the currently available anti-eosinophil biologics, the effect of eosinophil depletion should be monitored from the perspective of possible unwanted consequences., (© 2023 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Introduction: Very preterm birth is associated with lung function impairment later in life, but several aspects have not been studied. We aimed to comprehensively assess lung function at school age in very preterm infants and term controls, with special emphasis on bronchopulmonary dysplasia (BPD), sex, and bronchodilator response., Methods: At 12 years of age, 136 children born very preterm (85 with and 51 without BPD) and 56 children born at term performed spirometry, body plethysmography, impulse oscillometry, measurement of diffusion capacity, and multiple breath washout, before and after bronchodilator inhalation., Results: Airway symptoms and a diagnosis of asthma were more common in children born very preterm. These children had more airflow limitation, seen as lower forced expiratory volume in 1 s (FEV 1 ) (p < .001), FEV 1 /forced vital capacity (FVC) (p = .011), and mean forced expiratory flow between 25% and 75% of FVC (p < .001), and a higher total and peripheral airway resistance compared with term-born controls. There was no difference in total lung capacity but air trapping and lung clearance index were higher in children born very preterm. Diffusion capacity was lower in children born very preterm, especially in those with a diagnosis of BPD. In most other tests, the differences between preterm-born children with or without BPD were smaller than between children born preterm versus at term. Boys born preterm had more lung function deficits than preterm-born girls. In children born very preterm, airway obstruction was to a large extent reversible., Conclusion: At 12 years of age, children born very preterm had lower lung function than children born at term in most aspects and there was only little difference between children with or without BPD. Airway obstruction improved markedly after bronchodilator inhalation., (© 2023 The Authors. Pediatric Pulmonology published by Wiley Periodicals LLC.)

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Introduction: Chronic lung disorders involve pathological alterations in the lung tissue with hypoxia as a consequence. Hypoxia may influence the release of inflammatory mediators and growth factors including vascular endothelial growth factor (VEGF) and prostaglandin (PG)E 2 . The aim of this work was to investigate how hypoxia affects human lung epithelial cells in combination with profibrotic stimuli and its correlation to pathogenesis. Methods: Human bronchial (BEAS-2B) and alveolar (hAELVi) epithelial cells were exposed to either hypoxia (1% O 2 ) or normoxia (21% O 2 ) during 24 h, with or without transforming growth factor (TGF)-β1. mRNA expression of genes and proteins related to disease pathology were analysed with qPCR, ELISA or immunocytochemistry. Alterations in cell viability and metabolic activity were determined. Results: In BEAS-2B and hAELVi, hypoxia significantly dowregulated genes related to fibrosis, mitochondrial stress, oxidative stress, apoptosis and inflammation whereas VEGF receptor 2 increased. Hypoxia increased the expression of Tenascin-C, whereas both hypoxia and TGF-β1 stimuli increased the release of VEGF, IL-6, IL-8 and MCP-1 in BEAS-2B. In hAELVi, hypoxia reduced the release of fibroblast growth factor, epidermal growth factor, PGE 2 , IL-6 and IL-8, whereas TGF-β1 stimulus significantly increased the release of PGE 2 and IL-6. TGF-β1 stimulated BEAS-2B cells showed a decreased release of VEGF-A and IL-8, while TGF-β1 stimulated hAELVi cells showed a decreased release of PGE 2 and IL-8 during hypoxia compared to normoxia. Metabolic activity was significantly increased by hypoxia in both epithelial cell types. Discussion: In conclusion, our data indicate that bronchial and alveolar epithelial cells respond differently to hypoxia and profibrotic stimuli. The bronchial epithelium appears more responsive to changes in oxygen levels and remodelling processes compared to the alveoli, suggesting that hypoxia may be a driver of pathogenesis in chronic lung disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Berggren-Nylund, Ryde, Löfdahl, Ibáñez-Fonseca, Kåredal, Westergren-Thorsson, Tufvesson and Larsson-Callerfelt.)

Introduction: Allergic asthmatics with both an early (EAR) and a late allergic reaction (LAR) following allergen exposure are termed 'dual responders' (DR), while 'single responders' (SR) only have an EAR. Mechanisms that differentiate DR from SR are largely unknown, particularly regarding the role and phenotypes of neutrophils. Therefore, we aimed to study neutrophils in DR and SR asthmatics., Methods: Thirty-four allergic asthmatics underwent an inhaled allergen challenge, samples were collected before and up to 24 h post-challenge. Cell differentials were counted from bronchial lavage, alveolar lavage and blood; and tissue neutrophils were quantified in immune-stained bronchial biopsies. Lavage neutrophil nuclei lobe segmentation was used to classify active (1-4 lobes) from suppressive neutrophils (≥5 lobes). Levels of transmigration markers: soluble (s)CD62L and interleukin-1Ra, and activity markers: neutrophil elastase (NE), DNA-histone complex and dsDNA were measured in lavage fluid and plasma., Results: Compared with SR at baseline, DR had more neutrophils in their bronchial airways at baseline, both in the lavage (p = .0031) and biopsies (p = .026) and elevated bronchial neutrophils correlated with less antitransmigratory IL-1Ra levels (r = -0.64). DR airways had less suppressive neutrophils and more 3-lobed (active) neutrophils (p = .029) that correlated with more bronchial lavage histone (p = .020) and more plasma NE (p = .0016). Post-challenge, DR released neutrophil extracellular trap factors in the blood earlier and had less pro-transmigratory sCD62L during the late phase (p = .0076) than in SR., Conclusion: DR have a more active airway neutrophil phenotype at baseline and a distinct neutrophil response to allergen challenge that may contribute to the development of an LAR. Therefore, neutrophil activity should be considered during targeted diagnosis and bio-therapeutic development for DR., (© 2022 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Objective: The aim of this study was to investigate if desquamated oral epithelial cells (DOECs) express the epidermal growth factor (EGF) and if these cells thereby may contribute to salivary EGF contents., Background: DOECs have recently been shown to harbor the antimicrobial peptide LL-37, proposing that they may also store other biologically important salivary peptides/proteins. The EGF peptide is a growth factor which plays a critical role to maintain epithelial integrity and promote epithelial healing. The EGF is produced by salivary glands, but it is not known whether DOECs contain the EGF and thereby contribute to salivary EGF levels., Materials and Methods: DOECs were isolated from unstimulated whole saliva collected from four healthy volunteers. EGF protein expression was determined in cell lysates by dot blot and ELISA. Cellular distribution of cytokeratin, the proliferation marker Ki67, and EGF immunoreactivity were assessed by immunocytochemistry. EGF gene expression was investigated by qPCR. Expression of EGF transcript and protein in DOECs was compared to that in the human cultured keratinocyte cell line (HaCaT) cells., Results: EGF protein expression was detected in DOEC cell lysates by both dot blot and ELISA. Strong cytoplasmic EGF immunoreactivity was observed in DOECs, although some cells showed only a weak immunoreactive signal for EGF. Moreover, DOECs, besides containing EGF protein, also expressed transcript for EGF. Interestingly, ELISA analysis revealed that EGF protein contents were higher in DOECs than in HaCaT cells. ELISA analysis also disclosed that EGF concentration was about 10 times higher in whole saliva compared to DOECs. EGF transcript expression was about 50% lower in HaCaT cells stimulated with high (10%) compared to low (0.1%) concentration of fetal bovine serum, representing growth-stimulated and growth-restricted conditions, respectively, implying that growth-stimulus exerts negative feedback on EGF gene activity in HaCaT cells., Conclusion: Here, we show for the first time that DOECs express the EGF, arguing that these cells contribute to salivary EGF contents and hence may play a role in gingival epithelial repair and wound healing., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Alexandra Aidoukovitch et al.)