RESUMO
The coronavirus disease 2019 (COVID-19) pandemic has reached an unprecedented level. There is a strong demand for diagnostic and serological supplies worldwide, making it necessary for countries to establish their own technologies to produce high-quality biomolecules. The two main viral antigens used for the diagnostics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) are the structural proteins spike (S) protein and nucleocapsid (N) protein. The spike protein of SARS-CoV-2 is cleaved into S1 and S2, in which the S1 subunit has the receptor-binding domain (RBD), which induces the production of neutralizing antibodies, whereas nucleocapsid is an ideal target for viral antigen-based detection. In this study, we designed plasmids, pcDNA3.1/S1 and pcDNA3.1/N, and optimized their expression of the recombinant S1 and N proteins from SARS-CoV-2 in a mammalian system. The RBD was used as a control. The antigens were successfully purified from Expi293 cells, with high yields of the S1, N, and RBD proteins. The immunogenic abilities of these proteins were demonstrated in a mouse model. Further, enzyme-linked immunosorbent assays with human serum samples showed that the SARS-CoV-2 antigens are a suitable alternative for serological assays to identify patients infected with COVID-19.
RESUMO
The immune response plays a critical role in the pathophysiology of SARS-CoV-2 infection ranging from protection to tissue damage and all occur in the development of acute respiratory distress syndrome (ARDS). ARDS patients display elevated levels of inflammatory cytokines and innate immune cells, and T and B cell lymphocytes have been implicated in this dysregulated immune response. Mast cells are abundant resident cells of the respiratory tract and are able to release different inflammatory mediators rapidly following stimulation. Recently, mast cells have been associated with tissue damage during viral infections, but their role in SARS-CoV-2 infection remains unclear. In this study, we examined the profile of mast cell activation markers in the serum of COVID-19 patients. We noticed that SARS-CoV-2-infected patients showed increased carboxypeptidase A3 (CPA3) and decreased serotonin levels in their serum when compared with symptomatic SARS-CoV-2-negative patients. CPA3 levels correlated with C-reactive protein, the number of circulating neutrophils, and quick SOFA. CPA3 in serum was a good biomarker for identifying severe COVID-19 patients, whereas serotonin was a good predictor of SARS-CoV-2 infection. In summary, our results show that serum CPA3 and serotonin levels are relevant biomarkers during SARS-CoV-2 infection. This suggests that mast cells and basophils are relevant players in the inflammatory response in COVID-19 and may represent targets for therapeutic intervention.
Assuntos
COVID-19/diagnóstico , Carboxipeptidases A/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/diagnóstico , Mastócitos/imunologia , SARS-CoV-2/isolamento & purificação , Serotonina/metabolismo , Biomarcadores/análise , COVID-19/complicações , COVID-19/metabolismo , COVID-19/virologia , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Mastócitos/patologia , Índice de Gravidade de DoençaRESUMO
Recently, neutrophil extracellular traps (NETs), three-dimensional structures formed of neutrophil enzymes such as neutrophil elastase (NE) and nuclear components (DNA), have been associated with progression in different types of cancer. However, data remain scarce in breast cancer. Thus, the aim of this study was to associate NETs with clinical stages of breast cancer. A prospective analysis was performed in 45 plasma samples of female patients with newly diagnosed breast cancer. NE-DNA complexes were evaluated by ELISA. Optical density was dichotomized at the median for comparisons (low and high levels of NE-DNA). The most frequent clinical stage was localized (n = 28, 62%) followed by regional (n = 13, 29%) and distant (n = 4, 9%). Higher levels of NE-DNA complexes were observed in regional and distant stages compared to localized disease (68% vs 32%, p = 0.034). No differences were observed when comparing other clinical characteristics between both groups. We demonstrated that the levels of NETs increase in proportion to the stage of the disease, observing higher levels of NE-DNA complexes in regional and metastatic disease, which coincides with the proposed mechanism by which cancer progression and metastasis might result from the formation of NETs.