Introduction: The aim of this study was to analyze biomarkers that might predict the severity and progression of the SARS-CoV-2 infection, both in the acute phase and after recovery. Methods: Unvaccinated patients infected with the original strain of COVID-19 requiring ward (Group 1, n = 48) or ICU (Group 2, n = 41) admission were included. At the time of admission (visit 1), a clinical history was acquired, and blood samples were obtained. One and six months after discharge from the hospital (visits 2 and 3, respectively), a clinical history, lung function tests, and blood samples were carried out. At visit 2, patients also underwent a chest CT scan. Different cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, G-CSF, GM-CSF, IFN-ɣ, MCP-1, MIP-1β, and TNF-α) and lung fibrosis biomarkers (YKL-40 and KL-6) were measured in blood samples obtained at visits 1, 2, and 3. Results: At visit 1, IL-4, IL-5, and IL-6 levels were higher in Group 2 (p = 0.039, 0.011, and 0.045, respectively), and IL-17 and IL-8 levels were higher in Group 1 (p = 0.026 and 0.001, respectively). The number of patients in Groups 1 and 2 who died during hospitalization was 8 and 11, respectively. YKL-40 and KL-6 levels were higher in patients who died. Serum YKL-40 and KL-6 levels determined at visit 2 correlated negatively with FVC (p = 0.022 and p = 0.024, respectively) and FEV1 (p = 0.012 and p = 0.032, respectively) measured at visit 3. KL-6 levels also correlated negatively with the diffusing capacity of the lungs for carbon monoxide (DLCO, p = 0.001). Conclusions: Patients who required ICU admission had higher levels of Th2 cytokines, while patients admitted to the ward showed an innate immune response activation, with IL-8 release and Th1/Th17 lymphocyte contribution. Increased levels of YKL-40 and KL-6 were associated with mortality in COVID-19 patients.

Bird-related hypersensitivity pneumonitis (BRHP) is an interstitial lung disease induced by avian proteins. The immunopathological pathways involved in the disease are still unknown. This study assesses the cellular immune response and the cytokine pattern in a mouse model of BRHP. On days −3 and −1, mice were intraperitoneally sensitized with commercial pigeon serum (PS) or saline. Intranasal instillations with PS or saline were carried out on three consecutive days/week over either 3 weeks (Group 1) or 12 weeks (Group 2). Leukocyte and cytokine patterns in lung tissue and pulmonary inflammation in bronchoalveolar lavage (BAL) were analysed. Both groups presented increases in resident monocytes, interstitial macrophages and type 2 dendritic cells (DCs), but also reductions in inflammatory monocytes, alveolar macrophages and tolerogenic DCs compared with their control groups. Group 1 had increased levels of eosinophils and T cells with reductions in neutrophils and B cells, while Group 2 showed high levels of B cells. Both groups exhibited increases in Th1 and Th2 cytokines. Group 2 also showed increased levels of IL-23, a Th17 cytokine. Increased levels of neutrophils, eosinophils and lymphocytes were observed in BAL samples of both groups compared with controls. In the first stages of BRHP, there is a mixed Th1/Th2 immune response, while during the progression of the disease, although there is a Th1 response, the cytokine levels seem to indicate a switch towards a Th2/Th17 mixed response.

Introduction: Bird-related hypersensitivity pneumonitis is an interstitial lung disease induced by avian proteins, specially pigeon antigens. Complete avoidance to inhaled antigens is an essential step to ameliorate respiratory symptoms and prevent disease progression. The aim of the present study was to develop a lateral flow immunoassay (LFIA) method to detect pigeon antigens in environmental air samples. Methods: Twenty-two different air samples were collected from two different dovecotes of the province of Barcelona with an automated air sampler. Pigeon allergens were mixed with labeled specific anti-pigeon antibodies. The immunocomplexes formed migrate through a nitrocellulose membrane and bound to a line of immobilized specific anti-pigeon antibodies giving a visible band. Strip density values were calculated and compared with the concentration determined by an already standardized ELISA method for pigeon antigens. LFIA strips results were also visually analysed by five independent evaluators. Results: LFIA assay was able to detect a range of concentrations from 420 to 3360 ng/ml of pigeon antigen. A strong correlation was observed between the concentration determined by the ELISA method and the relative density results of the LFIA (rs:0.935; p<0.0001). Bland-Altman plot also confirmed a good agreement between both methods (mean difference: -1.626; p<0.0001). A kappa index of 0.736 (p<0.0001) was obtained between the observers evaluating the strip results. Conclusions: A rapid and easy semiquantitative pigeon allergen estimation method was developed. There was a good correlation between results obtained with ELISA method and LFIA assay. Study funded by ISCIII (PI15 / 01954), FEDER and FUCAP.

Introduction: YKL-40 protein is a biomarker of fibrosis and tissue remodeling produced by macrophages, neutrophils and epithelial cells. KL-6 protein is a biomarker of interstitial lung diseases mainly secreted by type II pneumocytes. This study assesses whether YKL-40 and KL-6 levels may play a role as biomarkers in hypersensitivity pneumonitis (HP). Methods: YKL-40 and KL-6 levels were determined in serum and induced sputum samples from 60 patients diagnosed with HP and 122 healthy controls (22 smokers) using a commercial ELISA kit. ROC curves were used to determine sensibility and specificity of both biomarkers in evaluating progression to pulmonary fibrosis in HP patients. Results: YKL-40 levels (median and range) in serum and sputum samples of HP patients were 66 (20-744) and 129 (35-754) ng/ml, respectively. KL-6 levels were 822 (176-6962) and 459 (14-8325) UI/ml, respectively. No correlation was observed between YKL40 and KL-6 levels in serum and sputum. A cut-off level of 51.5 ng/ml for serum YKL-40 had a sensitivity of 76% and a specificity of 63%. For KL-6, a cut-off level of 348.2 UI/ml had a sensitivity of 81% and a specificity of 76%. Significant differences were found in KL-6 serum levels between healthy controls and HP patients (p<0.0001). For YKL-40 these differences were found in serum levels between healthy controls and < 60 years old HP patients (p<0.0001). Conclusions: YKL-40 and KL-6 are high in HP patients. KL-6 seems a better biomarker because has high sensitivity and specificity and is not modified by age. Longitudinal studies are necessary to establish the role of these biomarkers in the pathogenesis of HP. Study funded by ISCIII (PI15/01954), FEDER and FUCAP.

Introduction: Hypersensitivity pneumonitis (HP) is usually caused by the inhalation of avian and fungal proteins. The present study assesses a cohort of Urban Pest Surveillance and Control Service (UPSCS) workers with high exposure to avian and fungal antigens, in order to identify their degree of sensitization to these antigens and the potential risk of developing HP. Methods: Study population: bird investigators and/or managers at UPSCS of the Public Health Agency, Parks and Gardens staff in Barcelona and employees of private urban pest control firms. Workers were divided according to their work activity: Pruning and Others. All individuals underwent a medical interview regarding exposure, pulmonary function tests and specific IgG antibodies. Antigenic proteins of pigeon sera were investigated using 2-dimensional immunoblotting with sera from patients with HP, asymptomatic exposed controls and healthy volunteers. Results: 101 workers have been recruited to date (76 men, average age: 42 yrs); 41 in the Pruning group and 60 in the others group. In the Pruning group, specific parakeet IgGs were higher (p=0.03) and FVC% and DLCO/VA% were lower (p=0.04 and 0.01, respectively). Two-dimensional immunoblotting showed protein bands of 20-25 KDa recognised by HP patients but not by workers. Conclusions: A high degree of sensitization to avian and fungal antigens was observed in the study population. In the Pruning group there were workers with alterations in some pulmonary function parameters. We identified several proteins that may play a role in the development of pathological differences between HP patients and exposed workers. Study funded by ISCIII (PI15 / 01954), FEDER and FUCAP.

Introduction: Up to 40% of patients with hypersensitivity pneumonitis (HP) progress to pulmonary fibrosis, suffering a loss of lung function that can lead to death. The aim of this study was to determine the involvement of the immune response in the development of pulmonary fibrosis in patients with advanced HP. Methods: Cross-sectional study conducted in 19 patients with fibrotic HP and 10 patients with idiopathic pulmonary fibrosis (IPF) who underwent lung transplantation. Healthy donors (n = 8) were included as control group. Lung samples were obtained from Vall d’Hebron and Ciberes Biobanks. Cytokines with a Th1, Th2 and Th17 profile were determined in tissue homogenate supernatant and MUC16, biomarker of fibrosis progression, was analysed in paraffin-embedded lung tissue. Results: Both patients with fibrotic HP and IPF had higher levels of GM-CSF, IL-7, IL-13 and IL-23 compared to healthy donors. Fifty per cent of patients with fibrotic HP had a more pronounced Th2 profile with higher levels of IL-5 and IL-6. This group of patients also had increased levels of IL-1β, IL-8, G-CSF and MCP-1 compared to HP patients with a low Th2 profile. HP patients with an increased Th2 profile had higher levels of IL-7 and IL-17 while those with a low Th2 profile had higher levels of IL-2, IL-12 and IL-23 compared to healthy controls. Maximum expression of MUC16 was observed in patients with IPF. Conclusions: Both patients with fibrotic HP and IPF have increased levels of Th17-related cytokines. HP patients with a high Th2 profile have an increased activation of the adaptive immune response with high production of cytokines involved in the recruitment of granulocytes. Study funded by ISCII(PI18/00345), FEDER and FUCAP.