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For the treatment of severe COVID-19, supplementation with human plasma-purified α-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins (“spike”). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-α and IL-1β mRNA expression and protein release were significantly inhibited (by about 46–50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359-394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1-0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies. Funding: Alpha-1 Antitrypsin Laurell’s Training Award (ALTA) for supporting Srinu Tumpara. Sí

Oxidative stress is linked to the inflammation observed in asthma and COPD and might be involved in exacerbations. Both COPD and asthma exacerbations often involve rhinovirus (RV) infection. Impaired anti-viral signaling and attenuated viral clearance in the bronchial epithelium has been hypothesized for these diseases. However, it is not known whether oxidative stress affects the anti-viral signaling capacity. Bronchial epithelial cells from asthma and COPD donors were infected with RV or treated with the oxidative stressor H2O2 followed by exposure to the synthetic RV replication intermediate poly(I:C). Gene and protein levels of antioxidants and anti-viral responses were measured 3h and 24h post poly(I:C) exposure. RV infection and poly(I:C) stimulation induced protein levels of the antioxidants SOD1 and SOD2. poly(I:C) was used to ascertain a constant infection-like burden in the interaction experiments. Upon pre-treatment with increasing doses of H2O2 the antioxidant response to dsRNA was dose-dependently decreased. This data suggests exaggerated H2O2-induced oxidative stress. Further, poly(I:C)-induced IFNs gene expression was reduced after pre-treatment with H2O2. This was associated with a reduced expression of the pattern recognition receptors RIG-I, MDA5 and TLR3 both on gene and protein level. Knockdown of TLR3 but not RIG-I/MDA5 abrogated impairment of poly(I:C)-induced IFNs gene expression by H2O2. Oxidative stress impairs anti-viral signaling in asthma and COPD bronchial epithelium, likely by reducing RV-responsiveness of TLR3. These findings shed light on molecular mechanisms potentially causing reduced IFNs at exacerbation.

Cristina Esquinas,1,2 Sabina Janciauskiene,3 Ricardo Gonzalo,4 Gemma Mas de Xaxars,4 Beata Olejnicka,5 Irene Belmonte,6 Miriam Barrecheguren,1 Esther Rodriguez,1 Alexa Nuñez,1 Francisco Rodriguez-Frias,6 Marc Miravitlles1 1Pneumology Department, University Hospital Vall d’Hebron, CIBER de Enfermedades Respiratorias (CIBERES), Barcelona, 2Public Health, Mental, Maternal and Child Health Nursing Department, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain; 3Department of Respiratory Medicine, Hannover Medical School, BREATH, German Center for Lung Research (DZL), Hannover, Germany; 4Statistics and Bioinformatics Unit (UEB), Vall d’Hebron Research Institute (VHIR), Barcelona, Spain; 5Department of Experimental Medical Science, Lund University, Lund, Sweden; 6Biochemistry Department, University Hospital Vall d’Hebron, Barcelona, Spain Introduction: COPD has complex etiologies involving both genetic and environmental determinants. Among genetic determinants, the most recognized is a severe PiZZ (Glu342Lys) inherited alpha1-antitrypsin deficiency (AATD). Nonetheless, AATD patients present a heterogeneous clinical evolution, which has not been completely explained by sociodemographic or clinical factors. Here we performed the gene expression profiling of blood cells collected from mild and severe COPD patients with PiZZ AATD. Our aim was to identify differences in messenger RNA (mRNA) and microRNA (miRNA) expressions that may be associated with disease severity. Materials and methods: Peripheral blood mononuclear cells from 12 COPD patients with PiZZ AATD (6 with severe disease and 6 with mild disease) were used in this pilot, high-throughput microarray study. We compared the cellular expression levels of RNA and miRNA of the 2 groups, and performed functional and enrichment analyses using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene-ontology (GO) terms. We also integrated the miRNA and the differentially expressed putative target mRNA. For data analyses, we used the R statistical language R Studio (version 3.2.5). Results: The severe and mild COPD–AATD groups were similar in terms of age, gender, exacerbations, comorbidities, and use of augmentation therapy. In severe COPD–AATD patients, we found 205 differentially expressed genes (DEGs) (114 upregulated and 91 downregulated) and 28 miRNA (20 upregulated and 8 downregulated) compared to patients with mild COPD–AATD disease. Of these, hsa-miR-335-5p was downregulated and 12 target genes were involved in cytokine signaling, MAPK/mk2, JNK signaling cascades, and angiogenesis were much more highly expressed in severe compared with mild patients. Conclusions: Despite the small sample size, we identified downregulated miRNA (hsa-miR-335) and the activation of pathways related to inflammation and angiogenesis on comparing patients with severe vs mild COPD–AATD. Nonetheless, our findings warrant further validation in large studies. Keywords: alpha1-antitrypsin deficiency, COPD, gene expression, miRNAs, integrative analysis

Benjamin Waschki,1–3 Henrik Watz,2,3 Olaf Holz,4,5 Helgo Magnussen,2,3 Beata Olejnicka,6 Tobias Welte,5,7 Klaus F Rabe,1,3 Sabina Janciauskiene5,7 1Pneumology, LungenClinic Grosshansdorf, Grosshansdorf, Germany; 2Pulmonary Research Institute at LungenClinic Grosshansdorf, Grosshansdorf, Germany; 3Airway Research Center North (ARCN), German Center for Lung Research (DZL), Grosshansdorf, Germany; 4Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany; 5Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research (DZL), Hannover, Germany; 6Department of Medicine, Trelleborg Hospital, Trelleborg, Sweden; 7Department of Respiratory Medicine,Hannover Medical School, Hannover, Germany Introduction: Plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of fibrinolysis, is associated with thrombosis, obesity, insulin resistance, dyslipidemia, and premature aging, which all are coexisting conditions of chronic obstructive pulmonary disease (COPD). The role of PAI-1 in COPD with respect to metabolic and cardiovascular functions is unclear. Methods: In this study, which was nested within a prospective cohort study, the serum levels of PAI-1 were cross-sectionally measured in 74 stable COPD patients (Global Initiative for Chronic Obstructive Lung Disease [GOLD] Stages I–IV) and 18 controls without lung disease. In addition, triglycerides, high-density lipoprotein cholesterol, fasting plasma glucose, waist circumference, blood pressure, smoking status, high-sensitive C-reactive protein (hs-CRP), adiponectin, ankle–brachial index, N-terminal pro-B-type natriuretic peptide, and history of comorbidities were also determined. Results: The serum levels of PAI-1 were significantly higher in COPD patients than in controls, independent of a broad spectrum of possible confounders including metabolic and cardiovascular dysfunction. A multivariate regression analysis revealed triglyceride and hs-CRP levels to be the best predictors of PAI-1 within COPD. GOLD Stages II and III remained independently associated with higher PAI-1 levels in a final regression analysis. Conclusion: The data from the present study showed that the serum levels of PAI-1 are higher in patients with COPD and that moderate-to-severe airflow limitation, hypertriglyceridemia, and systemic inflammation are independent predictors of an elevated PAI-1 level. PAI-1 may be a potential biomarker candidate for COPD-specific and extra-pulmonary manifestations. Keywords: plasminogen activator inhibitor-1, chronic obstructive pulmonary disease, cardiovascular disease, inflammation, hypertriglyceridemia, ankle-brachial index, N-terminal pro-B-type natriuretic peptide

Background It has been observed that patients with allergic asthma/rhinitis have increased apoptosis of peripheral blood cells. This study was designed to explore the idea that the markers of apoptosis may help predict the response of allergen immunotherapy. Methods The Allergy Department of University Hospital, Malmö, Sweden, recruited a total of 58 young adults (Results In patients receiving immunotherapy plasma gelsolin levels were higher relative to those without immunotherapy (the median level was 23.97 μg/mL [range, 18–35.8 μg/mL] versus 21.2 μg/mL [range, 13.9–29.8 μg/mL]; p = 0.012) and were similar to those of healthy controls (24.7 μg/mL [range, 17.4–35.3 μg/mL]). Plasma levels of sFas, Fas-L, CCL17, and TIMP-1 did not differ between study groups. Only in controls did the plasma gelsolin levels inversely correlate to the levels of soluble Fas. Conclusion Allergen-specific immunotherapy increases plasma levels of gelsolin, an antioxidant and antiapoptotic protein.

There is strong evidence that tobacco smoke-induced COPD is associated with an altered redox balance in intracellular/extracellular compartments and an uncontrolled immune-inflammatory response in genetically predisposed individuals. Primary line antioxidants (superoxide dismutases, SODs, catalase, CAT) and the scavenging thioredoxin (TRX) system play pivotal roles in the maintenance of redox homeostasis. Here we have investigated wherever any relationship exists between the density of immunoreactivity to SODs/CAT/TRX and lymphoid aggregates (LAs), macrophages (MQ) or neutrophils infiltration in COPD. Methods: The micro-localizations of MQ, neutrophils, LAs, SODs, CAT, and TRX were investigated by immunocytochemistry using paraffin-embedded lung sections from non-smokers (n=10) and COPD patients (n=10). Histological/immunohistochemical analyses and quantification were performed using the same Region-of-Interest (ROI) in different anatomical compartments: small airway, peripheral pulmonary artery, and alveolar parenchyma. Results: The percentage of CuZn-SOD/TRX immunoreactivity was significantly higher (p In summary, the data suggest that these redox-regulating proteins are highly expressed in severe COPD and there is a relationship between the expression of up-down regulation of antioxidants and the local LAs, MQ, and neutrophils infiltration.

A split sample study was performed on 109 bronchial brushings and washings and evaluated from conventional slides (CS) and CytoRich Red/Tripath preparations (CRR/Tripath). Unassessable bronchial washings were significantly more frequent in CS (5 vs. 0), but as all brushings were assessable with both methods, no overall diagnostic advantage was found. CS and CRR/Tripath gave discordant diagnoses in one case with a final benign diagnosis and in six cases with final malignant diagnoses. In the benign case, atypia was assessed in CS. In the malignant cases, suspected malignancy was found in one CRR/Tripath and one CS, atypia vs. benign assessment was also balanced, with three atypias in CRR/Tripath and two in CS. The better preserved cells in CRR/Tripath facilitated correct diagnosis in some cases, but might also lead to false positive diagnoses. In small cell carcinomas diagnostic hints such as smearing and moulding were less pronounced in CRR/Tripath but this did not affect the diagnostic accuracy. Overall, the diagnostic performance with CRR/Tripath was at least as good as with conventional slides, although statistically no difference could be seen. The number of slides and screening time, and thereby cost was significantly reduced with CRR/Tripath, thus the liquid-based method is preferred for bronchial washings and brushings. Diagn. Cytopathol. 2013. © 2013 Wiley Periodicals, Inc. (Less)

A split sample study was performed on 109 bronchial brushings and washings and evaluated from conventional slides (CS) and CytoRich Red/Tripath preparations (CRR/Tripath). Unassessable bronchial washings were significantly more frequent in CS (5 vs. 0), but as all brushings were assessable with both methods, no overall diagnostic advantage was found. CS and CRR/Tripath gave discordant diagnoses in one case with a final benign diagnosis and in six cases with final malignant diagnoses. In the benign case, atypia was assessed in CS. In the malignant cases, suspected malignancy was found in one CRR/Tripath and one CS, atypia vs. benign assessment was also balanced, with three atypias in CRR/Tripath and two in CS. The better preserved cells in CRR/Tripath facilitated correct diagnosis in some cases, but might also lead to false positive diagnoses. In small cell carcinomas diagnostic hints such as smearing and moulding were less pronounced in CRR/Tripath but this did not affect the diagnostic accuracy. Overall, the diagnostic performance with CRR/Tripath was at least as good as with conventional slides, although statistically no difference could be seen. The number of slides and screening time, and thereby cost was significantly reduced with CRR/Tripath, thus the liquid-based method is preferred for bronchial washings and brushings.