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Coagulation activation is a prominent feature of SARS-CoV-2 infection (COVID-19). Activation of the contact system and intrinsic pathway has increasingly been implicated in the prothrombotic state observed in both sterile and infectious inflammatory conditions. We thus sought to assess activation of the contact system and intrinsic pathway in subjects with COVID-19 infection. Baseline plasma levels of protease:serpin complexes indicative of activation of the contact and intrinsic pathways were measured in samples from inpatients with COVID-19 and healthy individuals. Cleaved kininogen, a surrogate for bradykinin release, was measured by ELISA, while extrinsic pathway activation was assessed by microvesicle tissue factor-mediated factor Xa generation (MVTF). Samples were collected within 24 hours of COVID-19 diagnosis. Thirty patients with COVID-19 and thirty age- and sex-matched controls were enrolled. Contact system and intrinsic pathway activation in COVID-19 were demonstrated by increased plasma levels of factor XIIa:C1 esterase inhibitor (FXIIa:C1), kallikrein:C1, FXIa:C1, FXIa:1antitrypsin, and FIXa:antithrombin (FIXa:AT). MVTF levels were also increased in COVID-19 subjects. Since FIXa:AT levels were associated with both contact/intrinsic pathway complexes and MVTF, activation of FIX likely occurs through both the contact/intrinsic and extrinsic pathways. Among the protease:serpin complexes measured, FIXa:AT complexes were uniquely associated with clinical indices of disease severity, specifically the total length of hospitalization, length of ICU stay, and extent of lung CT changes. We conclude that the contact/intrinsic pathway may contribute to the pathogenesis of the prothrombotic state in COVID-19. Larger prospective studies are required to confirm whether FIXa:AT complexes are a clinically useful biomarker of adverse clinical outcomes.

Abstract(–)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell invasion, angiogenesis and metastasis. Herein, we evaluated the in vivo effects of EGCG in acute myeloid leukaemia (AML) using an acute promyelocytic leukaemia (APL) experimental model (PML/RARα). Haematological analysis revealed that EGCG treatment reversed leucocytosis, anaemia and thrombocytopenia, and prolonged survival of PML/RARα mice. Notably, EGCG reduced leukaemia immature cells and promyelocytes in the bone marrow while increasing mature myeloid cells, possibly due to apoptosis increase and cell differentiation. The reduction of promyelocytes and neutrophils/monocytes increase detected in the peripheral blood, in addition to the increased percentage of bone marrow cells with aggregated promyelocytic leukaemia (PML) bodies staining and decreased expression of PML-RAR oncoprotein corroborates our results. In addition, EGCG increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells; and the combination of all-trans retinoic acid (ATRA) plus EGCG yield higher increase the expression of CD15 marker. These findings could be explained by a decrease of peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) expression and reactive oxygen species (ROS) increase. EGCG also decreased expression of substrate oncoproteins for PIN1 (including cyclin D1, NF-κB p65, c-MYC, and AKT) and 67 kDa laminin receptor (67LR) in the bone marrow cells. Moreover, EGCG showed inhibition of ROS production in NB4 cells in the presence of N-acetyl-L-cysteine (NAC), as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. Furthermore, apoptosis of spleen cells was supported by increasing expression of BAD and BAX, parallel to BCL-2 and c-MYC decrease. The reduction of spleen weights of PML/RARα mice, as well as apoptosis induced by EGCG in NB4 cells in a dose-dependent manner confirms this assumption. Our results support further evaluation of EGCG in clinical trials for AML, since EGCG could represent a promising option for AML patient ineligible for current mainstay treatments.

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Green tea (GT) treatment was evaluated for its effect on the immune and antineoplastic response of elderly acute myeloid leukemia patients with myelodysplasia-related changes (AML-MRC) who are ineligible for aggressive chemotherapy and bone marrow transplants. The eligible patients enrolled in the study (n = 10) received oral doses of GT extract (1000 mg/day) alone or combined with low-dose cytarabine chemotherapy for at least 6 months and/or until progression. Bone marrow (BM) and peripheral blood (PB) were evaluated monthly. Median survival was increased as compared to the control cohort, though not statistically different. Interestingly, improvements in the immunological profile of patients were found. After 30 days, an activated and cytotoxic phenotype was detected: GT increased total and naïve/effector CD8+ T cells, perforin+/granzyme B+ natural killer cells, monocytes, and classical monocytes with increased reactive oxygen species (ROS) production. A reduction in the immunosuppressive profile was also observed: GT reduced TGF-β and IL-4 expression, and decreased regulatory T cell and CXCR4+ regulatory T cell frequencies. ROS levels and CXCR4 expression were reduced in bone marrow CD34+ cells, as well as nuclear factor erythroid 2–related factor 2 (NRF2) and hypoxia-inducible factor 1α (HIF-1α) expression in biopsies. Immune modulation induced by GT appears to occur, regardless of tumor burden, as soon as 30 days after intake and is maintained for up to 180 days, even in the presence of low-dose chemotherapy. This pilot study highlights that GT extracts are safe and could improve the immune system of elderly AML-MRC patients.

ARHGAP21 is a member of the RhoGAP family of proteins involved in cell growth, differentiation, and adhesion. We have previously shown that the heterozygous Arhgap21 knockout mouse model (Arhgap21+/−) presents several alterations in the hematopoietic compartment, including increased frequency of hematopoietic stem and progenitor cells (HSPC) with impaired adhesion in vitro, increased mobilization to peripheral blood, and decreased engraftment after bone marrow transplantation. Although these HSPC functions strongly depend on their interactions with the components of the bone marrow (BM) niche, the role of ARHGAP21 in the marrow microenvironment has not yet been explored. In this study, we investigated the composition and function of the BM microenvironment in Arhgap21+/− mice. The BM of Arhgap21+/− mice presented a significant increase in the frequency of phenotypic osteoblastic lineage cells, with no differences in the frequencies of multipotent stromal cells or endothelial cells when compared to the BM of wild type mice. Arhgap21+/− BM cells had increased capacity of generating osteogenic colony-forming units (CFU-OB) in vitro and higher levels of osteocalcin were detected in the Arhgap21+/− BM supernatant. Increased expression of Col1a1, Ocn and decreased expression of Trap1 were observed after osteogenic differentiation of Arhgap21+/− BM cells. In addition, Arhgap21+/− mice recipients of normal BM cells showed decreased leucocyte numbers during transplantation recovery. Our data suggest participation of ARHGAP21 in the balanced composition of the BM microenvironment through the regulation of osteogenic differentiation.

Nuclear factor-erythroid 2 related factor (NRF2) is involved in cell defense and survival against endogenous and exogenous stress. Constitutively active Nrf2 in malignant cells increases the expression of cytoprotective genes and consequently, enhances proliferation via metabolic reprograming and inhibition of apoptosis (Leinonem, Advances in cancer Research, 2014). NRF2 is persistently activated in many human tumors, including acute myelogenous leukemia thus, inhibition of NRF2 activity may be a promising strategy for leukemia therapy. Flavonoids, present in vegetables, fruit and propolis, might exert antitumoral effects through induction of apoptosis and chromatin remodeling (Link, Biochem Pharmacol., 2010). A previous study from our group showed that Quercetin (Qu), a natural polyphenolic flavonoid compound, induced apoptosis, partly due to its DNA demethylating activity, through HDAC inhibition and by the enrichment of H3ac and H4ac in the promoter regions of genes involved in the apoptosis pathway, leading to their transcription activation (Alvarez, Clinical epigenetics 2018). In the present study, we evaluated the effect of Qu as a modulator of NRF2. This study was performed in vivo in human xenograft acute myeloid leukemia (AML) models, and in vitro using leukemia cell lines. Qu treatment (50 µM Qu) for 48h downregulated HDAC4, NRF2 and p-NRF2 at protein levels (p<0.05; p<0.005; p<0.005 respectively). Imaging Flow Cytometry (AMNIS, ImageStream ISX mkIITM) and Confocal Microscopy evidenced a decrease in NRF2 nuclear localization. Furthermore, combined treatment with the proteasome inhibitor MG132 prevented degradation of NRF2, indicating that treatment increased proteasomal degradation. Loss of NRF2 decreases HDAC4, a redox sensitive histone deacetylase, resulting in an increased expression of miR-1 and miR-206 (Singh, J Clin Invest. 2013). Herein, expression profile of 84 miRNAs (Apoptosis miRNA PCR array) were performed in samples from human xenograft model. Treatment up-regulated the expression profile of 5% (n=4) of the 84 miRNAs evaluated, corresponding exclusively to miRNAs that target anti-apoptotic genes and to miRNAs that have been demonstrated to have pro-apoptotic functions. Furthermore, expression levels of miR-1, miR-133a/b, which target anti-apoptotic genes and miR-206, a pro apoptotic miR, were validated in xenograft model samples, resulting in a significant up-regulation of the expression levels in treated animals compared to controls (p<0.05). In addition, lentiviral sh down regulation of NRF2, led to an increased apoptosis, decreased cell survival and an up regulation of miRNA 206 expression in Qu treated cells. In summary, Qu might induce programmed cell death in part, by decreasing NRF2 nuclear localization, by inducing NRF2 proteasomal degradation and down regulation of HDAC4 which led to up-regulation of pro apoptotic miRNAs. Disclosures No relevant conflicts of interest to declare.