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INTRODUCTION: Glioblastoma (GBM) is the most common and deadliest primary brain tumor, characterized by chemoradiation resistance and an immunosuppressive tumor microenvironment (TME). SARS-CoV-2, the COVID-19 virus, produces a significant proinflammatory response and a spectrum of clinical presentations after central nervous system infection. METHOD(S): Patient-derived GBM tissue, primary cell lines, and organoids were analyzed with immunohistochemistry and pixel-line intensity quantification. Data from tumor-bulk and single-cell transcriptomics served to describe the cell-specific expression of SARS-CoV-2 receptors in GBM and its association with the immune TME phenotype. Normal brain and iPSC-derived organoids served as controls. RESULT(S): We demonstrate that patient-derivedGBMtissue and cell cultures express SARS-CoV2 entry factors such as ACE2, TMPRSS2, and NRP1. NRP1 expression was higher in GBM than in normal brains (p<0.05), where it plays a crucial role in SARS-CoV-2 infection. NRP1 was expressed in a cell-type and phenotype-specific manner and correlated with TME infiltration of immunosuppressive cells: M2 macrophages (r = 0.229), regulatory T cells (r = 0.459), NK cells (r = -0.346), and endothelial cells (r = 0.288) (p < 0.05). Furthermore, gene ontology enrichment analysis showed that leukocyte migration and chemotaxis are among the top 5 biological functions mediated by NRP1 (p < 0.05). We found our GBM organoids recapitulate tumoral expression of SARSCoV- 2 entry factors, which varies based on distance from surface as surrogate of TME oxygenation (p < 0.05). CONCLUSION(S): GBM cancer cells and immune TME cells express SARS-CoV-2 entry factors. Glioblastoma organoids recapitulate this expression and allow for currently undergoing studies analyzing the effect of SARS-CoV-2 infection in GBM. Our findings suggest that SARSCoV- 2 could potentially target GBM, opening the door to future studies evaluating SARS-CoV-2-driven immune modulation.
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Background: Uncontrolled systemic infammation characterizes COVID-19 and autoinfammatory diseases such as adult-onset Still's disease (AOSD). Biosynthesis of pro-resolving mediators (SPMs), i.e. lipoxins (LX), resolvins (Rv), pro-tectins (PD), and maresins (MaR), ensures infammation shutdown and tissue repair, limiting neutrophils recruitment and stimulating macrophages to remove apoptotic cells. Among protectins, reduction of PD1 was found in the lungs of mice infected with the H5N1 infuenza virus and experimental treatment with PD1 resulted in increased animals' survival (Morita M et al 2013). Objectives: We investigated the effects of SPMs in pathogenesis and clinical evolution of AOSD and compared these data with mild and severe COVID-19. Finally, we analyzed the potential role of PD1 in modulating the infammatory response of macrophages obtained from AOSD patients, COVID-19 patients and healthy donors (HDs). Methods: 21 patients hospitalized for COVID-19 (10 ICU and 11 hospitalized in medical clinical unit) and 13 patients with AOSD were enrolled. Plasma PD1 levels from patients and controls were analyzed by ELISA, and mono-cytes-derived macrophages were polarized into M1 and M2 phenotype. We analyzed the effect of PD-1 on macrophages differentiation. At 10 days, macrophages were analyzed for surface expression of subtypes markers by flow cytometry. Cytokines production was measured in supernatants by Bio-Plex Assays. Peripheral blood mononuclear cells (PBMCS) from 3 AOSD patients, 2 COVID-19 patients and 3 HDs were obtained. Next-generation deep sequencing was then performed to identify the differences in PBMCs transcripts profiles. Results: AOSD patients with systemic scored (SS) ≥1 showed an increase of PD1 levels compared to AOSD patients with lower systemic score (p=0.04) (Figure 1A). Similarly, plasma levels of PD1 were increased in COVID-19 patients independently from their clinical subsets, compared to HDs (p=0.02). In vitro treatment with PD1 of monocytes-derived macrophages from AOSD and COVID-19 patients induced a signifcant increase of M2 polarization vs control (p<0.05) (Figure 1B). Furthermore, a signifcant release of IL-10 and CCL4 from M2 macrophages was observed when compared to control (p<0.05) (Figure 1C). In the transcriptomes from 3 AOSD patients (2 mild and 1 severe), 2 COVID-19 patients (1 mild and 1 ICU) and 2 HDs, we observed that genes involved in infammation, lipid catabolism and monocytes activation were spe-cifcally dysregulated in AOSD and COVID-19 patients when compared to HDs. Among them pla2g15, pla2g12a, pla2g2d, involved in mobilization of SPMs precursors, were signifcant upregulated in patients compared to HDs (p<.01, ;log2FoldChange;>1.2) (Figure 1D). The largest part of the genes involved in infammation, lipid catabolism, and monocytes activation are less expressed in AOSD patients when compared to COVID19 patients, as reported in Table 1. Conclusion: The counterbalance by SPMs during infammation is still a largely unexplored pathway. Our study suggests that an imbalance of SPMs in autoin-fammatory diseases as well as COVID-19. The modulation of SPMs as observed in our experiments, might represent a new possible therapeutic strategy during AOSD and COVID-19.
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Solid tumors are characterized by extensive immune suppressive inflammation, vascular leak, fibrosis and organ damage. Similarly, SARS-CoV-2 infections induce aberrant pulmonary and systemic inflammation, vascular leak, coagulation, fibrosis and fatal organ damage. We previously demonstrated that macrophages in solid tumors strongly expressed phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinately controls granulocyte and monocyte trafficking to tumors as well as wound-healing-type macrophage transcription in cancer and fibrosis. We also observed that macrophages in COVID-19 lungs strongly expressed PI3Kγ. To identify therapeutic strategies to suppress COVID-19-associated inflammation, we characterized lung tissue of COVID19 patients using multiplex immunohistochemistry and tissue transcriptomics. Lungs of deceased patients exhibited substantial infiltration by neutrophils and wound-healing macrophages, fibrosis and alveolar type II cell depletion. In animal models of lung inflammation, bacterial infections, viral infection and SARS-CoV-2 infection, PI3Kγ deletion or inhibition with the cancer therapeutic IPI-549 (eganelisib) suppressed pulmonary and systemic inflammation, reduced lung damage, and promoted survival. These studies demonstrate the essential role of PI3Kγ in inflammatory diseases as well as cancer and support the use of PI3Kγ inhibitors such as eganelisib to suppress inflammation and promote survival in pulmonary infections like SARS-CoV-2 and cancer.
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Vitamin D [25 (OH)D] plays a role in many of biological processes, such as bone metabolism, immunomodulation, cell proliferation, differentiation, and regulation. Also, it has anti-inflammatory, antifibrotic, and antioxidant effects. Due to the immunomodulatory effects of 25 (OH)D, its deficiency is blamed for a higher risk for COVID-19 infection. Serum concentrations of 25 (OH)D were inversely associated with proinflammatory cytokines such as increased IL-6, CRP levels, and increased risk of pneumonia or ARDS. Lower 25 (OH)D concentrations are associated with a higher risk for infections, especially from the respiratory tract [1]. Chronic vitamin D deficiency can induce the renin-angiotensin system activation and leads to fibrotic changes that can cause lung injury by inducing proinflammatory cytokine production in human monocytes/macrophages (2). Increased frequency of COVID-19 infection at high latitudes and worse prognosis of these cases made clinicians to think that 25 (OH)D levels may affect the risk and prognosis of COVID-19 infection [3]. In previous reports, in the early pandemic, a higher prevalence of vitamin D deficiency has been reported to be related to high rates of COVID-19 infection, higher risk of invasive mechanical ventilation (IMV), and mortality [6]. Whilst, it is reported that 25 (OH)D may not protect against COVID-19 infection in recent studies. Moreover, it was not associated with disease severity or lethality [4-6]. The active form of vitamin D binds to its receptor (VDR) and modulates its responses. VDR is located on chromosome 12q13, consisting of 9 exons. Vitamin D-VDR signaling regulates the expression of a wide range of physiological functions. Herein, VDR polymorphisms cause a dysfunctional receptor that affects VDR activity. Both innate and adaptive immune responses can vary according to different polymorphisms of VDR. Also VDR polymorphisms have been previously found to be associated with bacterial infections such as tuberculosis [7] and severe Respiratory Syncytial Virus (RSV) bronchiolitis in respect to vitamin D deficiency [8]. Moreover, it was demonstrated that different VDR polymorphisms such as FokI, BsmI, ApaI, and TaqI could change the course of RSV infection in several studies, respectively [8-10]. This study aimed to evaluate if there is any association between the VDR gene polymorphism at FokI, TaqI, BsmI, and ApaI alleles and the prognosis of COVID-19 in respect to vitamin D deficiency. Two-hundred ninety-seven (n=297) patients with reverse-transcription polymerase chain reaction (RT-PCR)-confirmed COVID-19 who were admitted to Marmara University Education and Research Hospital between April and October 2020 were enrolled. The severity of COVID-19 patients was classified into 1-10 according to WHO criteria. The patients' requirement for noninvasive mechanical ventilation (NIMV) or reservoir mask, their requirement for admission to intensive care unit (ICU), mortality, and WHO clinical progression scales were reviewed. Four variant regions of vitamin D receptor (VDR);FokI, BsmI, ApaI, and TaqI were determined using the Restriction Fragment Length Polymorphism (RFLP) technique. To conclude;The effect of VDR polymorphisms on the receptor function causes intensive care unit treatment, disease severity and mortality differences among patients with covid-19 infection in the clinical set-up. VDR Ff genotype was related with disease severity, TT with disease severity and aa with mortality respectively. As a result we have detected that 25 (OH)D levels were not related to COVID-19 infection severity and mortality. Additionally, it indicated that VDR polymorphisms are independently associated with the severity of COVID-19 and the survival of patients. More extensive studies are needed to determine the impact of polymorphisms on COVID-19 and explain the underlying cause.