Background: SARS-CoV-2 infected patients show heterogeneous clinical presentations ranging from mild symptoms to severe respiratory failure and death. Consequently, various markers reflect this wide spectrum of disease presentations. Methods: Our pilot cohort included moderate (n = 10) and severe (n = 10) COVID-19 patients, and 10 healthy controls. We determined plasma levels of nine acute phase proteins (APPs) by nephelometry, and full-length (M65), caspase-cleaved (M30) cytokeratin 18, and ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif 13) by ELISA. In addition, we examined whole plasma N-glycosylation by capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). Results: When compared to controls, COVID-19 patients had significantly lower concentrations of ADAMTS13 and albumin (ALB) but higher M30, M65, α1-acid glycoprotein (AGP), α1-antitrypsin (AAT), ceruloplasmin (CP), haptoglobin (HP), and high-sensitivity C-reactive protein (hs-CRP). The concentrations of α1-antichymotrypsin (ACT), α2-macroglobulin (A2MG) and serum amyloid A (SAA) proteins did not differ. We found significantly higher levels of AAT and M65 but lower ALB in severe compared to moderate COVID-19 patients. N-glycan analysis of the serum proteome revealed increased levels of oligomannose- and sialylated di-antennary glycans and decreased non-sialylated di-antennary glycan A2G2 in COVID-19 patients compared to controls. Conclusions: COVID-19-associated changes in levels and N-glycosylation of specific plasma proteins highlight complexity of inflammatory process and grant further investigations.

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.

Alpha-1-Antitrypsin (AAT) is a protein of the SERPINA1 gene. A single amino acid mutation (Lys342Glu) results in an expression of misfolded Z-AAT protein, which has a high propensity to intra- and extra-cellular polymerization. Here, we asked whether levels of circulating Z-AAT polymers are associated with the severity of lung disease, liver disease, or both. We obtained cross sectional data from the Dutch part of the Alpha1 International Registry of 52 ZZ-AAT patients who performed a pulmonary function test and donated a blood sample on the same day. From the Alpha-1 Liver Aachen Registry, we obtained a cohort of 40 ZZ-AAT patients with available data on their liver function. The levels of plasma Z-AAT polymers were determined using a LG96 monoclonal antibody-based sandwich ELISA. In a Dutch cohort, the median plasma level of Z-AAT polymers of patients diagnosed for pulmonary disease was 947.5 µg/mL (733.6–1218 µg/mL (95% CI)), which did not correlate with airflow obstruction or gas transfer value. In the Alpha-1 liver patient cohort, the median polymer level was 1245.9 µg/mL (753–2034 µg/mL (95% CI)), which correlated with plasma gamma-glutamyl transferase (GGT, rs = 0.57, p = 0.001), glutamate dehydrogenase (GLDH, rs = 0.48, p = 0.002) and triglycerides (TG, rs = 0.48, p = 0.0046). A Wilcoxon rank test showed higher Z-AAT polymer values for the liver over the lung group (p < 0.0001). These correlations support a possible link between plasma Z-AAT polymers and the liver function.

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.

Intracellular lipid droplets (LDs) can accumulate in response to inflammation, metabolic stresses, and other physiological/pathological processes. Herein, we investigated whether spike proteins of SARS-CoV-2 induce LDs in human peripheral blood mononuclear cells (PBMCs) and in pulmonary microvascular endothelial cells (HPMECs). PBMCs or HPMECs were incubated alone or with endotoxin-free recombinant variants of trimeric spike glycoproteins (Alpha, Beta, Delta, and Omicron, 12 µg/mL). Afterward, cells were stained with Oil Red O for LDs, cytokine release was determined through ELISA, and the gene expression was analyzed through real-time PCR using TaqMan assays. Our data show that spikes induce LDs in PBMCs but not in HPMECs. In line with this, in PBMCs, spike proteins lower the expression of genes involving lipid metabolism and LD formation, such as SREBF1, HMGCS1, LDLR, and CD36. On the other hand, PBMCs exposed to spikes for 6 or 18 h did not increase in IL-1β, IL-6, IL-8, MCP-1, and TNFα release or expression as compared to non-treated controls. Thus, spike-induced LD formation in PBMCs seems to not be related to cell inflammatory activation. Further detailed studies are warranted to investigate in which specific immune cells spikes induce LDs, and what are the pathophysiological mechanisms and consequences of this induction in vivo.

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.