Unraveling Links between Chronic Inflammation and Long COVID: Workshop Report.

As COVID-19 continues, an increasing number of patients develop long COVID symptoms varying in severity that last for weeks, months, or longer. Symptoms commonly include lingering loss of smell and taste, hearing loss, extreme fatigue, and "brain fog." Still, persistent cardiovascular and respiratory problems, muscle weakness, and neurologic issues have also been documented. A major problem is the lack of clear guidelines for diagnosing long COVID. Although some studies suggest that long COVID is due to prolonged inflammation after SARS-CoV-2 infection, the underlying mechanisms remain unclear. The broad range of COVID-19's bodily effects and responses after initial viral infection are also poorly understood. This workshop brought together multidisciplinary experts to showcase and discuss the latest research on long COVID and chronic inflammation that might be associated with the persistent sequelae following COVID-19 infection.

Stability and antioxidant activity of gossypol derivative immobilized on N-polyvinylpyrrolidone.

The objective of this study is analysis of stability and antioxidant and antiradical activities of the gossypol derivative - megosin conjugated with N-polyvinylpyrrolidone (PVP). The results of study have shown the greater stability of megosin+PVP than megosin in aqueous solution of wide range of pH. Here we also demonstrated that megosin+PVP, named rometin, possess high antioxidant activity in the same range as well known antioxidant trolox as determined by its ability to scavenge free ABTS(+) and DPPH radicals in vitro. In addition, megosin+PVP was able to prevent accumulation of products of lipid peroxidation (thiobarbituric acid reactive substances and diene conjugates) and lysophospholipids formation in mitochondria membranes caused by CCl(4)-induced oxidative stress in rat liver in vivo. Furthermore, megosin+PVP rescued mitochondrial functions, such as respiration and oxidative phosphorylation, which declined after CCl(4) administration. Thus we present that the conjugation of megosin to PVP increase its stability and remain antioxidant activity in vivo and in vitro.

Silencing of the CHM gene alters phagocytic and secretory pathways in the retinal pigment epithelium.

PURPOSE: Choroideremia (CHM) is an X-linked progressive degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid caused by mutations in the CHM gene, which encodes Rab escort-protein-1 (REP-1). REP-1 enables posttranslational isoprenyl modification of Rab GTPases, proteins that control vesicle formation, movement, docking, and fusion. The aim of this study was to determine the effect of REP-1 depletion on vesicular trafficking in phagocytic and secretory pathways of human RPE. METHODS: In vitro, REP-1 expression was inhibited in human fetal RPE (hfRPE) cells by siRNA knockdown and its effects measured on the uptake of bovine photoreceptor outer segments (POS), proteolysis of POS rhodopsin, phagosomal pH, phagosome fusion with early and late endosomes/lysosomes, and polarized secretion of cytokines. RESULTS: Depletion of REP-1 in human RPE cells did not affect POS internalization but reduced phagosomal acidification and delayed POS protein clearance. REP-1 depletion also caused a decrease in the association of POS-containing phagosomes with late endosomal markers (Rab7, LAMP-1) and increases in the secretion of monocyte chemotactic protein (MCP-1) and interleukin (IL)-8 by hfRPE cells. CONCLUSIONS: Lack of REP-1 protein expression in hfRPE cells leads to reduced degradation of POS most likely because of the inhibition of phagosome-lysosome fusion events and increased constitutive secretion of MCP-1 and IL-8. These observations may explain the accumulation of unprocessed outer segments within the phagolysosomes of RPE cells and the presence of inflammatory cells in the choroid of patients with CHM.

The immobilization of gossypol derivative on N-polyvinylpyrrolidone increases its water solubility and modifies membrane-active properties.

The conjugate of the gossypol derivative megosin (1) with N-polyvinylpyrrolidone named rometin (2) was synthesized. The effects of 1 and 2 on the structure and permeability of human erythrocytes and rat liver mitochondria were compared. Compound 1 induced dose-dependent erythrocyte hemolysis and increased mitochondrial permeability, with concomitant changes in membrane structure as determined by ESR and fluorescence anisotropy methods. Immobilization of 1 on N-polyvinylpyrrolidone (compound 2) increased its water solubility and reduced the intensity of its effects on erythrocyte membrane integrity and mitochondrial permeability, which correlated with a decrease in the membranes structural changes induced by the compound. Although the same concentrations of free and N-polyvinylpyrrolidone bound 1 were used, far less (14)C-labeled 1 was incorporated into the membranes from complex than free 1. The increase in water solubility and the reduction of membrane-active properties of 1 after immobilization on N-polyvinylpyrrolidone could explain our previous observation of the decreased toxicity of 1.

Late outgrowth endothelial progenitor cells in patients with age-related macular degeneration.

PURPOSE: To evaluate the feasibility of isolating and expanding endothelial progenitor cells (EPCs), in the form of late outgrowth endothelial progenitor cells (OECs), from the peripheral blood of an aged population, particularly patients affected by different forms of AMD. METHODS: Peripheral blood mononuclear cells were collected from young control subjects (n = 18) and from elderly subjects with non-AMD/low-risk dry AMD (n = 15), high-risk dry AMD (n = 6), or neovascular AMD (nvAMD; n = 32); cultured in established conditions; and observed for appearance of OEC clusters and growth characteristics on expansion. Expression of VEGF receptor-2 (KDR) in OECs after expansion was determined by Western blot. Plasma samples of study subjects were analyzed for CRP and VEGF levels. RESULTS: OEC cultures were successfully generated from a similar number of subjects in each group. After adjustment for all other variables, subjects with high-risk dry AMD had a 5.6-fold higher number of OEC clusters per 20 mL blood, and subjects with nvAMD had a 5.1-fold high number than did subjects with non-AMD/low-risk dry AMD (P < 0.05). High-risk dry AMD generated 63 times more (NS) and nvAMD 32-times more (P < 0.05) OECs on expansion of clusters than did non-AMD/low-risk dry AMD. Population doubling occurred significantly faster in cultures from nvAMD eyes compared to non-AMD/low-risk dry AMD eyes. In addition, a significant correlation between the number of OEC clusters, expanded OECs and levels of KDR was demonstrated. CONCLUSIONS: An OEC population was isolated and expanded from the blood of elderly control and AMD-affected patients and demonstrated significantly higher number of initial OEC clusters and expansion potential of OECs in patients at risk for or already affected by nvAMD. OECs may be used for further phenotypic, genetic, and functional analyses in patients with nvAMD.

RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo.

PURPOSE: To determine whether plasma low-density lipoprotein (LDL) could be internalized by the RPE and which receptors may be involved. A secondary objective was to determine whether ARPE19 cells could be used as a model to investigate cholesterol processing in the RPE. METHODS: Commercially available human LDL was labeled with rhodamine or AlexaFluor 568. Immunofluorescence was performed using commercially available antibodies to LDL-R, CD36, and LOX-1. Cells and tissues were imaged using epifluorescence and confocal fluorescence microscopy. Immunoblot analysis and RT-PCR were performed using published techniques. RESULTS: Intravenously injected rhodamine-labeled LDL (rhoLDL) was detected in the rat RPE by fluorescence confocal microscopy 24 hours after injection. The rhoLDL was present in some areas and absent in others. Cultured ARPE19 cells were also found to internalize LDL and oxidized LDL (oxLDL) readily. Using AlexaFluor 568-labeled LDL we determined that the average cultured RPE cell could internalize approximately 12 to 16 pg of LDL and oxLDL in 24 hours. Immunoblots readily detected the presence of CD36 and LDL-R in the cultured RPE cells but not LOX-1, whereas RT-PCR detected mRNA for all three receptors. Dual-labeling experiments using AlexaFluor 568-labeled LDL and AlexaFluor 488 for the immunolocalization of the receptors showed colocalization of LDL-R with the internalized LDL and CD36 with oxLDL particles. CONCLUSIONS: Plasma LDL readily enters the RPE through the choriocapillaris but is not found homogeneously throughout the retina. This may suggest some form of regulation to the permeability of the fenestrated choroidal endothelial cell junctions. ARPE19 cells are a good model for studying the internalization mechanisms of LDL and oxLDL in vitro. LDL may be used as a vector to carry hydrophobic molecules into the RPE.