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Background:: Hydroxyurea (HU) is a chemotherapeutic agent that reduces ribonucleotide reductase, stops DNA synthesis and repar, and therefore causes cell proliferation inhibition and apoptosis. Due to its cytostatic properties, HU is frequently used for treatment of myeloproliferative neoplasms, ovarian cancer, and sickle cell anemia. Nitric oxide (NO), produced by nitric oxide synthase (NOS) enzymes is a potent signaling molecule involved in blood flow regulation, neutrotransmission, and immunity. Although HU treatment increases NO levels, up to date it is not clear whether it originates from activation of NOS enzymes or HU degradation. Aims: The aim of this study was to determine the involvement of NOS2 enzyme in the cytostatic effect of HU. Methods: To examine the involvement of the NOS2 enzyme in the molecular mechanism of HU, we treated erythroleukemic HEL92.7.1 cells with pan-selective NOS inhibitor L-NAME (200µM, 1mM, and 5mM), NOS2 specific inhibitor 1400W (1, 10, and 100µM), or NOS2/NOS3 inhibitor DPI (1, 5, and 10µM), in combination with hydroxyurea (200µM), and monitored their effect on proliferation and cell cycle. Immunocytochemistry for the proliferation marker Ki67 was performed to assess proliferation, while cell distribution in cell cycle phases was determined by flow cytometry after propidium iodide staining. Colony forming assay have been performed with the bone marrow cells of Nos2 null mice after oral HU treatment to corroborate the data obtained by enzymatic inhibition. Results: In this study, we demonstrated that treatment of HEL92.7.1 cells with HU induces a dose-dependent increase in NOS2 protein levels and two products of the enzyme NOS - NO and citrulline. HU-induced citrulline levels can be reduced by treatment with the NOS inhibitor L-NAME, indicating that NO is produced de novo by the NOS enzyme rather than HU degradation. Inhibition of the NOS2 enzyme by L-NAME, 1400W, or DPI was sufficient to abolish HU-mediated inhibition of prolifer

The severity and mortality of coronavirus disease 2019 (COVID-19) are greater in males than in females, though the infection rate is the same in the two sexes. We investigated sex hormone differences associated with the hyperinflammatory immune response to SARS-CoV-2 on the basis of patients’ cytokine profiles and vaccination statuses. Clinical and laboratory data of 117 patients with COVID-19 were collected to examine sex differences associated with oxidative stress markers, neutrophil extracellular traps (NETs), and plasma cytokine levels up to 5 months from hospital admission. The testosterone and free testosterone levels were low in male patients with COVID-19 and returned to normal values after recovery from the disease. The dihydrotestosterone (DHT) levels were transiently reduced, while the sex hormone-binding globulin levels were decreased in post-COVID-19 male patients. The levels of the inflammatory cytokines interleukin-6 (IL-6) and IL-10 appeared generally increased at diagnosis and decreased in post-COVID-19 patients. In females, the concentration of tumor necrosis factor-alpha was increased by four times at diagnosis. The levels of the coagulation markers intercellular adhesion molecule-1 (ICAM-1) and E-selectin were consistently upregulated in post-COVID-19 female patients, in contrast to those of vascular cell adhesion molecule-1 (VCAM-1), P-selectin, and chemokine IL-8. DHT increased the levels of reactive oxygen species in the neutrophils of male patients, while estradiol decreased them in females. Markers for NET, such as circulating DNA and myeloperoxidase, were significantly more abundant in the patients’ plasma. Sex hormones have a potential protective role during SARS-CoV-2 infection, which is weakened by impaired testosterone synthesis in men.

Chronic inflammation is characterized by the production of reactive oxygen species (ROS), reactive nitrogen species, and inflammatory cytokines in myeloproliferative neoplasms (MPNs). In addition to these parameters, the aim of this study was to analyze the influence of ROS on the pro-liferation-related AKT/mTOR signaling pathway and the relationship with inflammatory factors in chronic myelogenous leukemia (CML). The activity of the antioxidant enzymes superoxide dis-mutase, glutathione peroxidase, and catalase is reduced in erythrocytes while levels of the oxidative stress markers malondialdehyde and protein carbonyl are elevated in the plasma of patients with CML. In addition, nitrogen species (nitrotyrosine, iNOS, eNOS) and inflammation markers (IL-6, NFkB, and S100 protein) were increased in granulocytes of CML while anti-inflammatory levels of IL-10 were decreased in plasma. CML granulocytes exhibited greater resistance to cytotoxic H2O2 activity compared to healthy subjects. Moreover, phosphorylation of the apoptotic p53 protein was reduced while the activity of the AKT/mTOR signaling pathway was increased, which was further enhanced by oxidative stress (H2O2) in granulocytes and erythroleukemic K562 cells. IL-6 caused oxidative stress and DNA damage that was mitigated using antioxidant or inhibition of inflammatory NFkB transcription factor in K562 cells. We demonstrated the presence of oxidative and ni-trosative stress in CML, with the former mediated by AKT/mTOR signaling and stimulated by in-flammation.

The COVID-19 pandemic has had a strong impact on people’s quality of life (QoL), which is affected by social and economic changes as well as by mental and physical health. The aim of this study was to determine QoL in post-COVID-19 patients who had required hospitalization, and to identify relevant sociodemographic data. We used questionnaires which considered demographic and socioeconomic data, health and vaccination status, the pandemic situation, and EQ-5D scoring. The interactions of all data and the scores of EQ-5D were analyzed. Multivariate logistic regression analysis was applied to the five dimensions of EQ-5D. In this single-hospital-cohort study, the average times elapsed since initial diagnosis and hospital admission were 2.5 (76.3 ± 18.1 days) and 5 months (155.4 ± 33.9 days), respectively. Post-COVID-19 females were 3–5 times more likely to be affected in terms of anxiety/depression, and in negative impact upon their usual activities, at 5 months after diagnosis. At the same time, reductions in mobility were 3–4 times more likely in elderly post-COVID-19 patients, whose levels of pain and discomfort increased. Single patients, those with low incomes, and those with severe clinical outcomes were 2–4 times more likely to experience a reduction in their usual activities, while the presence of co-morbidities and lower levels of education were associated with increased pain and discomfort. Aging-induced pain/discomfort and anxiety/depression were significantly exacerbated in elderly patients with widespread vaccination. Our study revealed effects of demographic and socioeconomic factors upon lower QoL in post-COVID-19 patients in four dimensions of EQ-5D: mobility, usual activity, pain/discomfort, and anxiety/depression, 5 months after first diagnosis and hospitalization.

Background. A vascular system inflammation is a risk of venous thromboembolism and can result in widespread microangiopathy with microvascular thrombosis. Methods. We performed laboratory clinical follow-up of patients with COVID-19 to compare gender differences. To study the sex difference in COVID-19 outcome we will measure estradiol and androgens: dihydrotestosterone (DHT) and sex hormone binding globulin (SHBP) in plasma of 63 COVID-19 patients, analyzed by ELISA. Their levels will be correlated to the adhesion molecules: soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), sE-selectin, and sP-selectin as biomarkers for inflammation and thrombosis. Results. DHT was increased (1.9 fold) in male COVID-19 patients compared to healthy male volunteers. SHBP was significantly increased in COVID-19 patients compared to healthy volunteers (p<0.05) as well as female vs. male COVID-19 patients (p<0.001, 2.5 fold). sVCAM-1 and sICAM-1 were increased in female COVID-19 patients compared to male COVID-19 patients and female volunteers, respectively (p<0.05). The sP-selection was significantly (p<0.01) increased in male vs. female COVID-19 patients. SHBP was in negative correlation with sP-selectin (p<0.05). DHT was in positive correlation with sVCAM-1 (p<0.05). Ferritin had 3-fold higher levels in male than female COVID-19 patients (p<0.001). Conclusions. Upregulation of androgen hormones and thrombotic biomarkers in COVID-19 patients demonstrate sex dependence.

Background: Chronic inflammation has been recognized in neoplastic disorders, including myeloproliferative neoplasm (MPN), as an important regulator of angiogenesis. Aims: We investigated the influence of vascular endothelial growth factor (VEGF) and pro-inflammatory interleukin-6 (IL-6) on the expression of angiogenic factors, as well as inflammation-related signaling in mononuclear cells (MNC) of patients with MPN and JAK2V617F positive human erythroleukemic (HEL) cells. Results: We found that IL-6 did not change the expression of angiogenic factors in the MNC of patients with MPN and HEL cells. However, IL-6 and the JAK1/2 inhibitor Ruxolitinib significantly increased angiogenic factors—endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor-1 alpha (HIF-1α)—in patients with polycythemia vera (PV). Furthermore, VEGF significantly increased the expression of HIF-1α and eNOS genes, the latter inversely regulated by PI3K and mTOR signaling in the MNC of primary myelofibrosis (PMF). VEGF and inhibitors of inflammatory JAK1/2, PI3K, and mTOR signaling reduced the eNOS protein expression in HEL cells. VEGF also decreased the expression of eNOS and HIF-1α proteins in the MNC of PMF. In contrast, VEGF increased eNOS and HIF-1α protein expression in the MNC of patients with PV, which was mediated by the inflammatory signaling. VEGF increased the level of IL-6 immunopositive MNC of MPN. In summary, VEGF conversely regulated gene and protein expression of angiogenic factors in the MNC of PMF, while VEGF increased angiogenic factor expression in PV mediated by the inflammation-related signaling. Conclusion: The angiogenic VEGF induction of IL-6 supports chronic inflammation that, through positive feedback, further promotes angiogenesis with concomitant JAK1/2 inhibition.