The efficacy of therapeutic plasma exchange in COVID-19 patients on endothelial tightness in vitro is hindered by platelet activation.

Coronavirus disease (COVID)-19 is characterised in particular by vascular inflammation with platelet activation and endothelial dysfunction. During the pandemic, therapeutic plasma exchange (TPE) was used to reduce the cytokine storm in the circulation and delay or prevent ICU admissions. This procedure consists in replacing the inflammatory plasma by fresh frozen plasma from healthy donors and is often used to remove pathogenic molecules from plasma (autoantibodies, immune complexes, toxins, etc.). This study uses an in vitro model of platelet-endothelial cell interactions to assess changes in these interactions by plasma from COVID-19 patients and to determine the extent to which TPE reduces such changes. We noted that exposure of an endothelial monolayer to plasmas from COVID-19 patients post-TPE induced less endothelial permeability compared to COVID-19 control plasmas. Yet, when endothelial cells were co-cultured with healthy platelets and exposed to the plasma, the beneficial effect of TPE on endothelial permeability was somewhat reduced. This was linked to platelet and endothelial phenotypical activation but not with inflammatory molecule secretion. Our work shows that, in parallel to the beneficial removal of inflammatory factors from the circulation, TPE triggers cellular activation which may partly explain the reduction in efficacy in terms of endothelial dysfunction. These findings provide new insights for improving the efficacy of TPE using supporting treatments targeting platelet activation, for instance.

Inflammatory profile of convalescent plasma to treat COVID: Impact of amotosalen/UVA pathogen reduction technology.

Blood products in therapeutic transfusion are now commonly acknowledged to contain biologically active constituents during the processes of preparation. In the midst of a worldwide COVID-19 pandemic, preliminary evidence suggests that convalescent plasma may lessen the severity of COVID-19 if administered early in the disease, particularly in patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms. This study examined the influence of photochemical Pathogen Reduction Treatment (PRT) using amotosalen-HCl and UVA light in comparison with untreated control convalescent plasma (n= 72 - paired samples) - cFFP, regarding soluble inflammatory factors: sCD40L, IFN-alpha, IFN-beta, IFN-gamma, IL-1 beta, IL-6, IL-8, IL-10, IL-18, TNF-alpha and ex-vivo inflammatory bioactivity on endothelial cells. We didn't observe significant modulation of the majority of inflammatory soluble factors (8 of 10 molecules tested) pre- or post-PRT. We noted that IL-8 concentrations were significantly decreased in cFFP with PRT, whereas the IL-18 concentration was increased by PRT. In contrast, endothelial cell release of IL-6 was similar whether cFFP was pre-treated with or without PRT. Expression of CD54 and CD31 in the presence of cFFP were similar to control levels, and both were significant decreased in when cFFP had been pre-treated by PRT. It will be interesting to continue investigations of IL-18 and IL-8, and the physiopathological effect of PRT- treated convalescent plasma and in clinical trials. But overall, it appears that cFFP post-PRT were not excessively pro-inflammatory. Further research, including a careful clinical evaluation of CCP-treated patients, will be required to thoroughly define the clinical relevance of these findings.

Inflammatory markers and auto-Abs to type I IFNs in COVID-19 convalescent plasma cohort study.

BACKGROUND: COVID-19 convalescent plasma (CCP) contains neutralising anti-SARS-CoV-2 antibodies that may be useful as COVID-19 passive immunotherapy in patients at risk of developing severe disease. Such plasma from convalescent patients may also have additional immune-modulatory properties when transfused to COVID-19 patients. METHODS: CCP (n = 766) was compared to non-convalescent control plasma (n = 166) for soluble inflammatory markers, ex-vivo inflammatory bioactivity on endothelial cells, neutralising auto-Abs to type I IFNs and reported adverse events in the recipients. FINDINGS: CCP exhibited a statistically significant increase in IL-6 and TNF-alpha levels (0.531 ± 0.04 vs 0.271 ± 0.04; (95% confidence interval [CI], 0.07371-0.4446; p = 0.0061) and 0.900 ± 0.07 vs 0.283 ± 0.07 pg/mL; (95% [CI], 0.3097-0.9202; p = 0.0000829) and lower IL-10 (0.731 ± 0.07 vs 1.22 ± 0.19 pg/mL; (95% [CI], -0.8180 to -0.1633; p = 0.0034) levels than control plasma. Neutralising auto-Abs against type I IFNs were detected in 14/766 (1.8%) CCPs and were not associated with reported adverse events when transfused. Inflammatory markers and bioactivity in CCP with or without auto-Abs, or in CCP whether or not linked to adverse events in transfused patients, did not differ to a statistically significant extent. INTERPRETATION: Overall, CCP exhibited moderately increased inflammatory markers compared to the control plasma with no discernible differences in ex-vivo bioactivity. Auto-Abs to type I IFNs detected in a small fraction of CCP were not associated with reported adverse events or differences in inflammatory markers. Additional studies, including careful clinical evaluation of patients treated with CCP, are required in order to further define the clinical relevance of these findings. FUNDING: French National Blood Service-EFS, the Association "Les Amis de Rémi" Savigneux, France, the "Fondation pour la Recherche Médicale (Medical Research Foundation)-REACTing 2020".

Inflammatory profile of convalescent plasma to treat COVID: Impact of amotosalen/UVA pathogen reduction technology

Blood products in therapeutic transfusion are now commonly acknowledged to contain biologically active constituents during the processes of preparation. In the midst of a worldwide COVID-19 pandemic, preliminary evidence suggests that convalescent plasma may lessen the severity of COVID-19 if administered early in the disease, particularly in patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms. This study examined the influence of photochemical Pathogen Reduction Treatment (PRT) using amotosalen‐HCl and UVA light in comparison with untreated control convalescent plasma (n= 72 – paired samples) - cFFP, regarding soluble inflammatory factors: sCD40L, IFN-alpha, IFN-beta, IFN-gamma, IL-1 beta, IL-6, IL-8, IL-10, IL-18, TNF-alpha and ex-vivo inflammatory bioactivity on endothelial cells. We didn’t observe significant modulation of the majority of inflammatory soluble factors (8 of 10 molecules tested) pre- or post-PRT. We noted that IL-8 concentrations were significantly decreased in cFFP with PRT, whereas the IL-18 concentration was increased by PRT. In contrast, endothelial cell release of IL-6 was similar whether cFFP was pre-treated with or without PRT. Expression of CD54 and CD31 in the presence of cFFP were similar to control levels, and both were significant decreased in when cFFP had been pre-treated by PRT. It will be interesting to continue investigations of IL-18 and IL-8, and the physiopathological effect of PRT- treated convalescent plasma and in clinical trials. But overall, it appears that cFFP post-PRT were not excessively pro-inflammatory. Further research, including a careful clinical evaluation of CCP-treated patients, will be required to thoroughly define the clinical relevance of these findings.

Comparison of bacterial filtration efficiency vs. particle filtration efficiency to assess the performance of non-medical face masks.

As a result of the current COVID-19 pandemic, the use of facemasks has become commonplace. The performance of medical facemasks is assessed using Bacterial Filtration Efficiency (BFE) tests. However, as BFE tests, require specific expertise and equipment and are time-consuming, the performance of non-medical facemasks is assessed with non-biological Particle Filtration Efficiency (PFE) tests which are comparatively easier to implement. It is necessary to better understand the possible correlations between BFE and PFE to be able to compare the performances of the different types of masks (medical vs. non-medical). In this study BFE results obtained in accordance with the standard EN 14683 are compared to the results of PFE from a reference test protocol defined by AFNOR SPEC S76-001 with the aim to determine if BFE could be predicted from PFE. Our results showed a correlation between PFE and BFE. It was also observed that PFE values were higher than BFE and this was attributed to the difference in particle size distribution considered for efficiency calculation. In order to properly compare these test protocols for a better deduction, it would be interesting to compare the filtration efficiency for a similar granulometric range.

Evaluation of in vitro activity of copper gluconate against SARS-CoV-2 using confocal microscopy-based high content screening.

CONTEXT: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged late in 2019 is the etiologic agent of coronavirus disease 2019 (Covid-19). There is an urgent need to develop curative and preventive therapeutics to limit the current pandemic and to prevent the re-emergence of Covid-19. This study aimed to assess the in vitro activity of copper gluconate against SARS-CoV-2. METHODS: Vero E6 cells were cultured with or without copper gluconate 18-24 hours before infection. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Infected cells were incubated in fresh medium containing varying concentration of copper gluconate (supplemented with bovine serum albumin or not) for an additional 48 -h period. The infection level was measured by the confocal microscopy-based high content screening method. The cell viability in presence of copper gluconate was assessed by XTT and propidium iodide assays. RESULTS: The viability of Vero E6 cells exposed to copper gluconate up to 200 µM was found to be similar to that of unexposed cells, but it dropped below 70 % with 400 µM of this agent after 72 h of continuous exposure. The infection rate was 23.8 %, 18.9 %, 20.6 %, 6.9 %, 5.3 % and 5.2 % in cells treated prior infection with 0, 2, 10, 25, 50 and 100 µM of copper gluconate respectively. As compared to untreated cells, the number of infected cells was reduced by 71 %, 77 %, and 78 % with 25, 50, and 100 µM of copper gluconate respectively (p < 0.05). In cells treated only post-infection, the rate of infection dropped by 73 % with 100 µM of copper gluconate (p < 0.05). However, the antiviral activity of copper gluconate was abolished by the addition of bovine serum albumin. CONCLUSION: Copper gluconate was found to mitigate SARS-CoV-2 infection in Vero E6 cells but this effect was abolished by albumin, which suggests that copper will not retain its activity in serum. Furthers studies are needed to investigate whether copper gluconate could be of benefit in mucosal administration such as mouthwash, nasal spray or aerosols.