High levels of pro-inflammatory SARS-CoV-2-specific biomarkers revealed by in vitro whole blood cytokine release assay (CRA) in recovered and long-COVID-19 patients.

BACKGROUND: Cytokines induced by SARS-CoV-2 infection play a crucial role in the pathophysiology of COVID-19 and hyperinflammatory responses have been associated with poor clinical outcomes, with progression to severe conditions or long-term subacute complications named as long-COVID-19. METHODS: In this cross-sectional study, we aimed to evaluate a set of antigen-specific inflammatory cytokines in blood from recovered COVID-19 individuals or who suffered a post-acute phase of SARS-CoV-2 infection compared to healthy individuals with no history of COVID-19 exposition or infection. Interferon-gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), tumor necrosis factor (TNF), IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, and IL-17A were quantified by multiplex cytometric bead assay and enzyme-linked immunosorbent assay after stimulation of whole blood with recombinant Spike protein from SARS-CoV-2. Additionally, all participants have evaluated for anti-(S) protein-specific IgG antibodies. Clinical specimens were collected within two months of COVID-19 diagnosis. RESULTS: A total of 47 individuals were enrolled in the study, a median age of 43 years (IQR = 14.5), grouped into healthy individuals with no history of infection or exposure to SARS-CoV-2 (unexposed group; N = 21); and patients from the Health Complex of the Rio de Janeiro State University (UERJ), Brazil, who were SARS-CoV-2 positive by RT-PCR (COVID-19 group)-categorized as recovered COVID-19 (N = 11) or long-COVID-19 (N = 15). All COVID-19 patients presented at least one signal or symptom during the first two weeks of infection. Six patients were hospitalized and required invasive mechanical ventilation. Our results showed that COVID-19 patients had significantly higher levels of IFN-γ, TNF, IL-1ß, IL-2, IL-6, IL-8, and IP-10 than the unexposed group. The long-COVID-19 group has presented significantly high levels of IL-1ß and IL-6 compared to unexposed individuals, but not from recovered COVID-19. A principal-component analysis demonstrated 84.3% of the total variance of inflammatory-SARS-CoV-2 response in the first two components, and it was possible to stratify IL-6, TNF, IL-1ß, IL-10, and IL-2 as the top-five cytokines which are candidates to discriminate COVID-19 group (including long-COVID-19 subgroup) and healthy unexposed individuals. CONCLUSION: We revealed important S protein-specific differential biomarkers in individuals affected by COVID-19, bringing new insights into the inflammatory status or SARS-CoV-2 exposition determination.

Influence of culture conditions on Vero cell propagation on non-porous microcarriers

Animal cell cultures are widely employed for the production of viral vaccines and for recombinant protein expression. The cell line Vero is a continuous, adherent cell line, which has been recommended by the World Health Organization for the production of human vaccines. For the large-scale production of vaccines, microcarriers, which are microspheres that serve as support for the cells, are being increasingly used. The use of microcarriers in stirred bioreactors allows high cell densities and, consequently, high virus titres to be achieved. With the aim of selecting appropriate culture conditions for the cultivation of Vero cells at high cell densities, in this work the influence of several variables (agitation rate, ratio of inoculated cells to microcarrier mass and fetal bovine serum concentration) on cell growth on Cytodex 1 microcarriers was studied. Under the best conditions determined, a comparison with Vero cell cultivation on Cytodex 3 microcarriers was carried out.

Lipase production by solid-state fermentation in fixed-bed bioreactors

O fungo Penicillium simplicissimum se mostrou, em trabalhos anteriores, um ótimo produtor de lipase por fermentação no estado sólido, quando cultivado em biorreatores do tipo bandeja, utilizando a torta de babaçu como meio de cultura. Com o objetivo de aumentar a produtividade e possibilitar uma ampliação de escala, foi investigado, no presente trabalho, o emprego de biorreatores de leito fixo com aeração forçada. Os biorreatores utilizados tinham 4 cm de diâmetro interno e 14 cm de altura útil. Empregando-se planejamento estatístico de experimentos como ferramenta, foram avaliadas as influências da temperatura e da vazão de ar sobre a produção de lipase nestes biorreatores. Os resultados obtidos permitiram ajustar um modelo empírico, o qual indicou que maiores atividades lipásicas são alcançadas para temperaturas mais baixas e vazões de ar mais altas. A atividade lipásica máxima (26,4 U/g) foi obtida para temperatura de 27°C e vazão de ar de 0,8 L/min.