Endotoxin tolerance and low activation of TLR-4/NF-κB axis in monocytes of COVID-19 patients.

Higher endotoxin in the circulation may indicate a compromised state of host immune response against coinfections in severe COVID-19 patients. We evaluated the inflammatory response of monocytes from COVID-19 patients after lipopolysaccharide (LPS) challenge. Whole blood samples of healthy controls, patients with mild COVID-19, and patients with severe COVID-19 were incubated with LPS for 2 h. Severe COVID-19 patients presented higher LPS and sCD14 levels in the plasma than healthy controls and mild COVID-19 patients. In non-stimulated in vitro condition, severe COVID-19 patients presented higher inflammatory cytokines and PGE-2 levels and CD14 + HLA-DRlow monocytes frequency than controls. Moreover, severe COVID-19 patients presented higher NF-κB p65 phosphorylation in CD14 + HLA-DRlow, as well as higher expression of TLR-4 and NF-κB p65 phosphorylation in CD14 + HLA-DRhigh compared to controls. The stimulation of LPS in whole blood of severe COVID-19 patients leads to lower cytokine production but higher PGE-2 levels compared to controls. Endotoxin challenge with both concentrations reduced the frequency of CD14 + HLA-DRlow in severe COVID-19 patients, but the increases in TLR-4 expression and NF-κB p65 phosphorylation were more pronounced in both CD14 + monocytes of healthy controls and mild COVID-19 patients compared to severe COVID-19 group. We conclude that acute SARS-CoV-2 infection is associated with diminished endotoxin response in monocytes. KEY MESSAGES: Severe COVID-19 patients had higher levels of LPS and systemic IL-6 and TNF-α. Severe COVID-19 patients presented higher CD14+HLA-DRlow monocytes. Increased TLR-4/NF-κB axis was identified in monocytes of severe COVID-19. Blunted production of cytokines after whole blood LPS stimulation in severe COVID-19. Lower TLR-4/NF-κB activation in monocytes after LPS stimulation in severe COVID-19.

Systemic redox imbalance in severe COVID-19 patients.

The aim of this study was to evaluate the systemic redox state and inflammatory markers in intensive care unit (ICU) or non-ICU severe COVID-19 patients during the hospitalization period. Blood samples were collected at hospital admission (T1) (Controls and COVID-19 patients), 5-7 days after admission (T2: 5-7 days after hospital admission), and at the discharge time from the hospital (T3: 0-72 h before leaving hospital or death) to analyze systemic oxidative stress markers and inflammatory variables. The reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) were analyzed in peripheral granulocytes and monocytes. THP-1 human monocytic cell line was incubated with plasma from non-ICU and ICU COVID-19 patients and cell viability and apoptosis rate were analyzed. Higher total antioxidant capacity, protein oxidation, lipid peroxidation, and IL-6 at hospital admission were identified in both non-ICU and ICU COVID-19 patients. ICU COVID-19 patients presented increased C-reactive protein, ROS levels, and protein oxidation over hospitalization period compared to non-ICU patients, despite increased antioxidant status. Granulocytes and monocytes of non-ICU and ICU COVID-19 patients presented lower MMP and higher ROS production compared to the healthy controls, with the highest values found in ICU COVID-19 group. Finally, the incubation of THP-1 cells with plasma acquired from ICU COVID-19 patients at T3 hospitalization period decreased cell viability and apoptosis rate. In conclusion, disturbance in redox state is a hallmark of severe COVID-19 and is associated with cell damage and death.

Alterations in CD39/CD73 axis of T cells associated with COVID-19 severity.

Purinergic signaling modulates immune function and is involved in the immunopathogenesis of several viral infections. This study aimed to investigate alterations in purinergic pathways in coronavirus disease 2019 (COVID-19) patients. Mild and severe COVID-19 patients had lower extracellular adenosine triphosphate and adenosine levels, and higher cytokines than healthy controls. Mild COVID-19 patients presented lower frequencies of CD4+ CD25+ CD39+ (activated/memory regulatory T cell [mTreg]) and increased frequencies of high-differentiated (CD27- CD28- ) CD8+ T cells compared with healthy controls. Severe COVID-19 patients also showed higher frequencies of CD4+ CD39+ , CD4+ CD25- CD39+ (memory T effector cell), and high-differentiated CD8+ T cells (CD27- CD28- ), and diminished frequencies of CD4+ CD73+ , CD4+ CD25+ CD39+ mTreg cell, CD8+ CD73+ , and low-differentiated CD8+ T cells (CD27+ CD28+ ) in the blood in relation to mild COVID-19 patients and controls. Moreover, severe COVID-19 patients presented higher expression of PD-1 on low-differentiated CD8+ T cells. Both severe and mild COVID-19 patients presented higher frequencies of CD4+ Annexin-V+ and CD8+ Annexin-V+ T cells, indicating increased T-cell apoptosis. Plasma samples collected from severe COVID-19 patients were able to decrease the expression of CD73 on CD4+ and CD8+ T cells of a healthy donor. Interestingly, the in vitro incubation of peripheral blood mononuclear cell from severe COVID-19 patients with adenosine reduced the nuclear factor-κB activation in T cells and monocytes. Together, these data add new knowledge to the COVID-19 immunopathology through purinergic regulation.

Influence of oregano essential oil on the inhibition of selected pathogens in "Alheira" during storage.

BACKGROUND: Plant-derived essential oils (EOs) have shown remarkable antimicrobial potential against spoil- age and pathogenic microorganisms in meat and meat products. The aim of this study was to investigate the influence of oregano EO on the inhibition of Salmonella Enteritidis, Listeria monocytogenes and Staphylo- coccus aureus in an internal mixture of “Alheira” during storage. METHODS: Different concentrations of oregano EO (4%, 1.5%, 0.5%, 0.195% and 0.0975%) were evaluated against the selected pathogens during 21 days of storage at 4°C. The pH and water activity values and lactic acid bacteria (LAB) counts were also evaluated. Finally, sensory assessment was performed. RESULTS: The antibacterial effect varied according to the oregano EO concentration used, and target pathogen. Oregano EO at 4% demonstrated the highest antimicrobial activity against all the pathogens tested. The low- est concentrations used (0.195% and 0.0975%) resulted in ~2–3 log reduction, but only for L. monocytogenes after 21 days of storage. Counts of LAB were ~109 CFU/ml for all samples and no differences in the pH and aw values were detected between samples. However, at a concentration of 0.195%, Oregano EO had a negative impact on consumer acceptance of “Alheira”. CONCLUSIONS: These results could be interesting for the meat industry, as a starting point for other studies that have now to concentrate on strategies to “mask” unpleasant sensorial alterations caused by EOs in “Alheira” and helping the industry to ensure the microbiological safety of its products.

Growth control of Listeria innocua 2030c on vacuum-packaged cold-smoked salmon by lactic acid bacteria.

Five bacteriocin-producing lactic acid bacteria (LAB): Enterococcus faecium ET05, Lactobacillus curvatus ET06, L. curvatus ET30, L. deldrueckii ET32 and Pediococcus acidilactici ET34, selected by their capacity for growth and producing inhibition in vitro at high salt-on-water content, low temperature and anaerobic atmosphere, conditions simulating cold-smoked fish, were inoculated onto salmon fillets, in co-culture with Listeria innocua 2030c, and cold-smoked processed (dry salted for 6 h; drying for 6 h; smoke for 2 h). The finished product was then packed under vacuum and stored at 5 degrees C. Enumeration of LAB and L. innocua was performed during storage. Results showed that strain E. faecium ET05 was the best biopreservative candidate for controlling L. innocua growth in vacuum-packaged cold-smoked salmon (CSS) processed under the salting/drying/smoking parameters referred above. L. curvatus ET30 and L. delbrueckii ET32 also showed a good biopreservation potential for CSS although they were less effective than the former. L. curvatus ET06 and P. acidilactici ET34 showed a bacteriostatic mode of action against the target bacteria in vitro as well as when inoculated into the salmon fillets. This study describes a potential application of five different LAB in the biopreservation of Listeria in CSS.

Could modifications of processing parameters enhance the growth and selection of lactic acid bacteria in cold-smoked salmon to improve preservation by natural means?

Several smoking conditions were examined with the objective of enhancing the numbers of lactic acid bacteria (LAB) by natural means in vacuum-packaged cold-smoked salmon during 21 days of storage at 5 degrees C. Three combinations of salting, drying, and smoking were used: (i) dry salting x time of salting (2 or 6 h); (ii) wet salting (6 h) x dry salting (6 h) x with or without sugar; and (iii) wet salting (6 h) x dry salting (6 h) x different times of smoking (2 or 6 h of drying and 2 or 6 h of smoking). Two batches were processed for each set of conditions. Determinations of pH and salt content in the water phase were carried out for products in each treatment. Microbiological analyses (total viable count, total LAB, Lactobacillus spp., and Enterobacteriaceae) also were conducted at the beginning of storage (t0) and after 21 days of refrigerated storage (tl). There were differential increases in total LAB and lactobacilli during the storage period according to the treatment performed. The most effective treatment to enhance LAB growth was 6 h of dry salting with sugar, 6 h of drying, and 2 h of smoking. These salting-drying-smoking conditions also selected the LAB as the dominant flora at the end of the storage period. The LAB promoted by these processing parameters seem to be potentially useful protective cultures because of their anti-Listeria activity. From the results of this research, we conclude that it is possible to enhance the growth of LAB in general and that of inhibitory strains in particular by suitable choices of processing parameters.