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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 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.

Neutrophils are an essential component of the innate immune response, but their prolonged activation can lead to chronic inflammation. Consequently, neutrophil homeostasis is tightly regulated through balance between granulopoiesis and clearance of dying cells. The bone marrow is both a site of neutrophil production and the place they return to and die. Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by the mutations in three types of molecular markers, with emphasis on Janus kinase 2 gene mutation (JAK2V617F). The MPN bone marrow stem cell niche is a site of chronic inflammation, with commonly increased cells of myeloid lineage, including neutrophils. The MPN neutrophils are characterized by the upregulation of JAK target genes. Additionally, MPN neutrophils display malignant nature, they are in a state of activation, and with deregulated apoptotic machinery. In other words, neutrophils deserve to be placed in the midst of major events in MPN. Our crucial interest in this review is better understanding of how neutrophils die in MPN mirrored by defects in apoptosis and to what possible extent they can contribute to MPN pathophysiology. We tend to expect that reduced neutrophil apoptosis will establish a pathogenic link to chronic inflammation in MPN.

Background: Oxidative stress is caused by imbalance between excessive production of reactive oxygen species and decreased capabilities of antioxidant system, and it is recognized as a feature in cancerogenesis, as well in hematologic malignancies. Previous studies have shown increasing expression of oxidative stress markers and antioxidant enzymes in lymph nodes progressing to aggressive lymphomas. Aims: The aim of our study was to assess the clinical and prognostic significance of oxidative stress markers in patients with untreated diffuse large B-cell lymphoma (DLBCL). Methods: We analysed 64 patients diagnosed with DLBLC during 2018 and 2019, while 27 healthy volunteers (51.9% males) served as a control group. The plasma sample and laboratory analyses were obtained prior to initiation of specific hematologic treatment. After completion of the therapy, the patients were followed up for up to 4 years and for each of them progression free survival (PFS) and overall survival (OS) were calculated. Malondialdehyde (MDA) and protein carbonyl (PC) were used as markers of oxidative stress in plasma of patients and volunteers, while catalase is used as an antioxidant marker. Results: The mean patients’ age was 56.2 years (range, 20–87); 51.6% were males. Majority of patients were analysed before 1L therapy (n=61; 95,3%), and had following clinical stages: Ann Arbor stage I 23.4%, stage II 37.5%, stage III 15.6% and stage IV 23.4%. Majority of the patients had satisfactory performance status (73.5% had ECOG PS ≥1), bulky tumorous mass was present in 34.4% of patients, whereas 70.3% had extranodal localisation of lymphoma. MDA (6.66±2.7 nmol/ml) was significantly increased (p<0.001, 2.6-fold), while PC (4.29±2.7 nmol/mg) was also significantly increased (p=0.0027, 5-fold) in patients with DLBCL compared to healthy volunteers. In opposite, antioxidant catalase (0.194±0.06 IU/ml) was significantly reduced (p=0.0034, 1.9-fold) in patients with DLBCL. MDA was in significant (p<0

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:: 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

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.

Hydroxyurea (HU) causes nitric oxide (NO) bioactivation, acting as both a NO donor and a stimulator of NO synthase (NOS). To examine whether HU effects are NO mediated by chemical degradation or enzymatic induction, we studied human and mouse erythroid cells during proliferation, apoptosis, and differentiation. The HU and NO donor demonstrated persisted versus temporary inhibition of erythroid cell growth during differentiation, as observed by γ-and β-globin gene expression. HU decreased the percentage of erythroleukemic K562 cells in the G2/M phase that was reversed by N-nitro l-arginine methyl ester hydrochloride (L-NAME). Besides activation of endothelial NOS, HU significantly increased apoptosis of K562 cells, again demonstrating NOS dependence. Administration of HU to mice significantly inhibited colony-forming unit-erythroid (CFU-E), mediated by NOS. Moreover, burst-forming-units-erythroid (BFU-E) and CFU-E ex vivo growth was inhibited by the administration of nitrate or nitrite to mice. Chronic in vivo NOS inhibition with L-NAME protected the bone marrow cellularity despite HU treatment of mice. NO metabolites and HU reduced the frequency of NOS-positive cells from CFU-E and BFU-E colonies that was reverted by NOS inhibition. HU regulation of the G2/M phase, apoptosis, differentiation, cellularity, and NOS immunoreactive cells was NOS dependent. Inhalation of NO therapy as well as strategies to increase endogenous NO production could replace or enhance HU activity.

In several systems, hydroxyurea has been shown to trigger nitric oxide (NO) release or activation of NO synthase (NOS). To elucidate this duality in its pharmacological effects, during myelosuppression, we individually examined hydroxyurea’s (NO releasing agent) and NO metabolites’ (stable NO degradation products) effects on erythroid colony growth and NOS/NO levels in mice using NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Hydroxyurea and nitrite/nitrate decreased the bone marrow cellularity that was blocked by PTIO only for the NO metabolites. Hydroxyurea inhibition of colony-forming unit-erythroid (CFU-E) formation and reticulocytes was reversed by PTIO. Moreover, hydroxyurea, through a negative feedback mechanism, reduced inducible NOS (iNOS) expressing cells in CFU-E, also prevented by PTIO. Nitrate inhibition of burst-forming units-erythroid (BFU-E) colony growth was blocked by PTIO, but not in mature CFU-E. The presented results reveal that NO release and/or production mediates the hydroxyurea inhibition of mature erythroid colony growth and the frequency of iNOS immunoreactive CFU-E.

common characteristic of malignancies is an increase of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In such circumstances macromolecules are damaged and the cell structure and function are disrupted. The oxidative DNA damage is considered to be the initial event in the developement of carcinogenesis. ROS affects the function of hematopoietic stem cells, changes the cell cycle progression, accelerаtes aging and induces hematological malignancies. The BCR/ABL oncogene and JAK2V617F mutation induces the formation of ROS in hematopoietic stem cells that leads to genomic instability and DNA damage. In the myeloproliferative neoplasm (MPN) patients, JAK2V617F mutation and BCR/ABL oncogene are associated with the constitutive activation of the PI3K/AKT/mTOR signaling pathway, which stimulates the growth and proliferation of cells. As ROS and RNS may affect the activity of mTOR, we have examined the parameters of antioxidant protection and oxidative damage of lipids and proteins in circulation as well as the AKT/mTOR signaling pathway activity in the MPN granulocytes and human erythroleukemia (HEL) cell line with JAK2V617F mutation. Methods: The study includes 110 de novo MPN patients: 30 polycythemia vera (PV), 24 essential thrombocythemia (ET), 34 primary myelofibrosis (PMF) as well as 22 patients with the diagnosis of chronic myeloid leukemia (CML) and 10 healthy subjects. Markers of oxidative and nitrosative stress in plasma and erythrocyte lysates were determined by the colorimetric method. The intracellular production of ROS was determined by fluorogenic reagent H2DCF-DA. The levels of nitrotyrosine, inducible NO synthase (iNOS) and the activation rate of AKT/mTOR/p70S6K kinases were determined in granulocytes of MPN patients using immunocytochemistry and immunoblotting methods. The influence of hydrogen peroxide (H2O2) and 2, 2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) oxidants on AKT/mTOR signal pathway, cell cycle and survival were te, Zajednička karakteristika maligniteta je povećana koncentracija reaktivnih vrsta kiseonika (ROS) i reaktivnih vrsta azota (RNS) koje mogu da prouzrokuju oštećenja makromolekula u ćeliji i dovedu do narušavanja njene strukture i funkcije. Oksidativno oštećenje DNK smatra se inicijalnim događajem u razvoju kancerogeneze. ROS utiču na funkciju hematopoetskih matičnih ćelija, dovode do promena ćelijskog ciklusa, ubrzanog starenja ćelija ili do nastanka hematoloških maligniteta. BCR/ABL onkogen i JAK2V617F mutacija indukuju stvaranje ROS u hematopoetskim matičnim ćelijama što vodi genomskoj nestabilnosti i oštećenju DNK. Pod uticajem JAK2V617F mutacije i BCR/ABL onkogena kod hroničnih mijeloproliferativnih neoplazmi (HMN) dolazi do konstitutivne aktivacije PI3K/AKT/mTOR signalnog puta, koji reguliše metabolizam i stimuliše rast i proliferaciju ćelija. S obzirom da ROS i RNS mogu uticati na aktivnost mTOR kinaze, ispitani su parametri antioksidativne zaštite i oksidativno izmenjenih lipida i proteina u cirkulaciji, kao i stepen aktivacije AKT/mTOR signalnog puta u granulocitima bolesnika sa HMN i humanoj eritroleukemijskoj ćelijskoj liniji (eng. Human erythroleukemia cell line, HEL) sa JAK2V617F mutacijom. Metode: U studiju je uključeno 110 de novo HMN bolesnika: 30 sa policitemijom verom (PV), 24 sa esencijalnom trombocitemijom (ET), 34 sa primarnom mijelofibrozom (PMF), 22 bolesnika sa dijagnozom hronične mijeloidne leukemije (HML) i 10 zdravih ispitanika. Markeri oksidativnog i nitrozativnog stresa u plazmi i lizatima eritrocita određeni su kolorimetrijskom metodom. Količina intracelularne produkcije ROS određena je upotrebom fluorogenog reagensa H2DCF-DA. Nivo nitrotirozina, inducibilne NO sintaze (iNOS) i stepen aktivacije AKT/mTOR/p70S6K kinaza određeni su u granulocitima bolesnika sa HMN imunocitohemijskom metodom i metodom imunoblota. U in vitro uslovima ispitan je uticaj vodonik peroksida (H2O2) i N- acetil cisteina na aktivaciju mTOR i ekspresiju iNOS i nitrotir

Purpose: A common feature of malignancies is increased reactive oxygen species (ROS) and reactive nitrogen species (RNS). We analyzed the influence of oxidative and nitrosative stress on the activation of AKT/mTOR signaling pathway in myeloproliferative neoplasms (MPN). Methods: Oxidative stress-induced gene expression in circulatory CD34(+) cells of MPN patients was studied by microarray analysis. Biomarkers of oxidative and nitrosative stress were determined using spectrophotometry in plasma and erythrocyte lysate. The levels of nitrotyrosine, inducible NO synthase (iNOS) and AKT/mTOR/p70S6K phosphorylation were determined by immunocytochemistry and immunoblotting in granulocytes of MPN patients. Results: Antioxidants superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPx1) gene expression were increased in circulatory CD34(+) cells, while SODI and GPx enzymes were reduced in the erythrocytes of MPN. Plasma malonyl-dialdehyde and protein carbonyl levels were elevated in MPN. The total antioxidant capacity in plasma and erythrocyte catalase (CT) activities was the most prominent in primary myelofibrosis (PMF) with JAK2V617F heterozygosity. The total nitrite/ nitrate (NOx) level was augmented in the plasma of PMF patients (p lt 0.001), while nitrotyrosine and iNOS were generally increased in the granulocytes of MPN patients. Activation of AKT/m TOR signaling was the most significant in PMF (p lt 0.01), but demonstrated JAK2V617F dependence and consequent p70S6K phosphorylation in the granulocytes of essential thrombocytemia (ET) and polycythemia vera (PV) patients. Hydrogen peroxide stimulated mTOR pathway, iNOS and nitrotyrosine quantities, the last one prevented by the antioxidant nacetyl-cysteine (NAC) in the granulocytes of MPN. Conclusion: Our study showed increased levels of oxidative and nitrosative stress parameters in MPN with JAK2V617F dependence. The ROS enhanced the constitutive activation of AKT/mTOR signaling and nitrosative parameters in MPN.

This study has been performed to determine the mechanism of activation of the myeloid related S100A proteins by inflammatory cytokines in myeloproliferative neoplasm (MPN). Besides microarray analysis of MPN-derived CD34(+) cells, we analysed the pro-inflammatory IL6 and anti-inflammatory IL10 dependence of NF-kappa B, PI3K-AKT, and JAK-STAT signalling during induction of S100A proteins in mononuclear cells of MPN, by immunoblotting and flow cytometry. We observed the reduced gene expression linked to NF-kappa B and inflammation signalling in MPN-derived CD34(+) cells. Both IL6 and IL10 reduced S100A8 and 100A9 protein levels mediated via NF-kappa B and PI3K signalling, respectively, in mononuclear cells of essential thrombocythemia (ET). We also determined the increased percentage of S100A8 and S100A9 positive granulocytes in ET and primary myelofibrosis, upgraded by the JAK2V617F mutant allele burden. S100A8/9 heterodimer induced JAK1/2-dependent mitotic arrest of the ET-derived granulocytes. Significance of the study We demonstrated that inflammation reduced the myeloid related S100A8/9 proteins by negative feedback mechanism in ET. S100A8/9 can be a diagnostic marker of inflammation in MPN, supported by the concomitant NF-kappa B and JAK1/2 signalling inhibition in regulation of myeloproliferation and therapy of MPN.

Hydroxyurea (HU) is a nonalkylating antineoplastic agent used in the treatment of hematological malignancies. HU is a DNA replication stress inducer, and as such, it may induce a premature senescence-like cell phenotype; however, its repercussion on bystander cell proliferation has not been revealed so far. Our results indicate that HU strongly inhibited peripheral blood mesenchymal stromal cells (PBMSC) proliferation by cell cycle arrest in S phase, and that, consequently, PBMSC acquire senescence-related phenotypical changes. HU-treated PBMSC display increased senescence-associated beta-galactosidase levels and p16(INK4) expression, as well as DNA damage response and genotoxic effects, evidenced by expression of gamma H2A.X and micronuclei. Moreover, HU-induced PBMSC senescence is mediated by increased reactive oxygen species (ROS) levels, as demonstrated by the inhibition of senescence markers in the presence of ROS scavenger N-acetylcysteine and NADPH oxidase inhibitor Apocynin. To determine the HU-induced bystander effect, we used the JAK2V617F-positive human erythroleukemia 92.1.7 (HEL) cells. Co-culture with HU-induced senescent PBMSC (HU-S-PBMSC) strongly inhibited bystander HEL cell proliferation, and this effect is mediated by both ROS and transforming growth factor (TGF)-beta expression. Besides induction of premature senescence, HU educates PBMSC toward an inhibitory phenotype of HEL cell proliferation. Finally, our study contributes to the understanding of the role of HU-induced PBMSC senescence as a potential adjuvant in hematological malignancy therapies.

Заједничка карактеристика малигнитета је повећана концентрација реактивних врста кисеоника (ROS) и реактивних врста азота (RNS) које могу да проузрокују оштећења макромолекула у ћелији и доведу до нарушавања њене структуре и функције. Оксидативно оштећење ДНК сматра се иницијалним догађајем у развоју канцерогенезе. ROS утичу на функцију хематопоетских матичних ћелија, доводе до промена ћелијског циклуса, убрзаног старења ћелија или до настанка хематолошких малигнитета. BCR/ABL онкоген и JAK2V617F мутација индукују стварање ROS у хематопоетским матичним ћелијама што води геномској нестабилности и оштећењу ДНК. Под утицајем ЈАК2V617F мутације и BCR/ABL онкогена код хроничних мијелопролиферативних неоплазми (ХМН) долази до конститутивне активације PI3K/AKT/mTOR сигналног пута, који регулише метаболизам и стимулише раст и пролиферацију ћелија. С обзиром да ROS и RNS могу утицати на активност mTOR киназе, испитани су параметри антиоксидативне заштите и оксидативно измењених липида и протеина у циркулацији, као и степен активације AKT/mTOR сигналног пута у гранулоцитима болесника са ХМН и хуманој еритролеукемијској ћелијској линији (енг. Human erythroleukemia cell line, HEL) са ЈАК2V617F мутацијом. Методе: У студију je укључено 110 de novo ХМН болесника: 30 са полицитемијом вером (ПВ), 24 са есенцијалном тромбоцитемијом (ЕТ), 34 са примарном мијелофиброзом (ПМФ), 22 болесника са дијагнозом хроничне мијелоидне леукемије (ХМЛ) и 10 здравих испитаника. Маркери оксидативног и нитрозативног стреса у плазми и лизатима еритроцита одређени су колориметријском методом. Количина интрацелуларне продукције ROS одређенa је употребом флуорогеног реагенса H2DCF-DA. Ниво нитротирозина, индуцибилне NO синтазе (iNOS) и степен активације АKT/mTOR/p70S6K киназа одређени су у гранулоцитима болесника са ХМН имуноцитохемијском методом и методом имуноблота. У in vitro условима испитан је утицај водоник пероксида (H2O2) и N- ацетил цистеина на активацију mТОR и експресију iNOS и нитротирозина. Ут, common characteristic of malignancies is an increase of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In such circumstances macromolecules are damaged and the cell structure and function are disrupted. The oxidative DNA damage is considered to be the initial event in the developement of carcinogenesis. ROS affects the function of hematopoietic stem cells, changes the cell cycle progression, accelerаtes aging and induces hematological malignancies. The BCR/ABL oncogene and JAK2V617F mutation induces the formation of ROS in hematopoietic stem cells that leads to genomic instability and DNA damage. In the myeloproliferative neoplasm (MPN) patients, JAK2V617F mutation and BCR/ABL oncogene are associated with the constitutive activation of the PI3K/AKT/mTOR signaling pathway, which stimulates the growth and proliferation of cells. As ROS and RNS may affect the activity of mTOR, we have examined the parameters of antioxidant protection and oxidative damage of lipids and proteins in circulation as well as the AKT/mTOR signaling pathway activity in the MPN granulocytes and human erythroleukemia (HEL) cell line with JAK2V617F mutation. Methods: The study includes 110 de novo MPN patients: 30 polycythemia vera (PV), 24 essential thrombocythemia (ET), 34 primary myelofibrosis (PMF) as well as 22 patients with the diagnosis of chronic myeloid leukemia (CML) and 10 healthy subjects. Markers of oxidative and nitrosative stress in plasma and erythrocyte lysates were determined by the colorimetric method. The intracellular production of ROS was determined by fluorogenic reagent H2DCF-DA. The levels of nitrotyrosine, inducible NO synthase (iNOS) and the activation rate of AKT/mTOR/p70S6K kinases were determined in granulocytes of MPN patients using immunocytochemistry and immunoblotting methods. The influence of hydrogen peroxide (H2O2) and 2, 2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) oxidants on AKT/mTOR signal pathway, cell cycle and survival were te

Purpose Previously, the family of S 100A proteins has been found to be associated with inflammation and myelopoiesis and to be able to induce or support myeloproliferation during chronic inflammation. Here, we studied the inflammatory myeloid-related proteins Si 00A4, S 100A8, S 100A9 and S100Al2 in myeloproliferative neoplasms (MPNs) in order to assess the involvement of chronic inflammation in the pathogenesis of MPN. Methods We analyzed the S100A4, S100A8, S100A9 and S100Al2 mRNA and protein levels in the bone marrow and circulation of 140 patients with MPN and 15 healthy controls using Western blotting, microarray-based mRNA expression profiling and ELISA assays, respectively. In addition we performed functional studies on the proliferation-related AKT and ERK1/2 signaling pathways in MPN-derived granulocytes using Western blotting and proteomic analyses. Results We found that the S100A mRNA levels were increased in MPN patient-derived circulatory CD34(+ )cells, and that their protein expression levels were also augmented in their granulocytes and bone marrow stroma cells, depending on the JAK2V617F mutation allele burden. We also found that calreticulin (CALR) mutations were related to reduced S100A8 plasma levels in primary myelofibrosis (PMF). The S100A8 plasma levels were found to be increased in MPN, the S100A9 plasma levels in PMF and essential thrombocythemia (ET), and the S100A12 plasma levels in polycythemia vera (PV). These 5100A plasma levels showed a positive correlation with the systemic inflammation marker IL-8, as well as with the numbers of leukocytes and thrombocytes, depending on the JAK2V617F mutation status. Additionally, we found that heterodimeric S100A8/9 can inhibit the AKT pathway in MPN-derived granulocytes mediated by the Toll-like receptor 4 (TLR4), depending on the CALR mutation status. Conversely, we found that blocking of the receptor for advanced glycation end products (RAGE) increased the S100A8/9-mediated inhibition of AKT signa

In accordance with increased proliferation in myeloproliferative neoplasm (MPN), the goal is to evaluate the immunoexpression of: beta-catenin, PPAR-gamma and Ki67 protein, to compare them with bone marrow ultrastructural characteristics in patients with MPN. Immunoexpression and electron microscopy of bone marrow was analyzed in 30 Ph-negative MPN patients, including per 10 patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The quantity of beta-catenin immunoreactive cells was significantly higher in PV then in ET (p lt 0.01) or PMF group of patients (p lt 0.01) and also in ET versus PMF group of patients (p lt 0.01). Erythroid lineage showed absent beta-catenin staining without immunoreactivity in nucleus. In contrast, immunoreactivity for PPAR-gamma was localized mostly in megakaryocytes and the highest number of PPAR-gamma immunopositive cells was detected in PMF group of patients. In addition, the proliferative Ki67 index was significantly increased in the PMF and PV patients compared to patients with ET. Also, the megakaryocytes showed abnormal maturation in PMF group of patients as determined by ultrastructural analysis. These results indicated that PV dominantly expressed beta-catenin and proliferation marker Ki67 in bone marrow, while PMF is linked preferentially to PPAR-gamma immunopositive megakaryocytes characterized by abnormal maturation.

Objective. Although it is known that nitric oxide modulates the activity of hypothalamic-pituitary-adrenal axis, its functional significance has not been elucidated. Additionally, there is no information on the effect of nitric oxide on cortical expression of glucocorticoid receptor. The purpose of this study was to investigate the influence of endogenous nitric oxide on structure and function of rat adrenal cortex and adrenocortical expression of glucocorticoid receptor. Methods. Adult female Wistar rats showing diestrus day 1 were treated with Nω-nitro-L-arginine-methyl ester (LNAME), an inhibitor of all three isoforms of nitric oxide synthase, at a dose of 30 mg/kg, subcutaneously. The concentrations of adrenocorticotropic hormone (ACTH) and corticosterone were determined by radioimmunoassay. Stereological and immunohistochemical analyses were performed on paraffin sections. Results. Blockade of nitric oxide production significantly increased blood levels of ACTH and corticosterone. Stereological analysis showed that treatment with L-NAME significantly decreased numerical density of the cells in all cortical zones. Consistent with the decreased numerical density, L-NAME significantly increased the volume of cells in cortical zones. Inhibition of nitric oxide synthesis decreased expression of glucocorticoid receptor in zona fasciculata and zona reticularis. Conclusion. Obtained results indicate that endogenous nitric oxide inhibits activity of adrenal cortex and modulates expression of glucocorticoid receptor., Cilj. Iako je poznato da azot-monoksid moduliše aktivnost hipotalamo-hipofizno-nadbubrežne osovine, funkcionalni značaj tog delovanja nije rasvetljen. Pored toga, dejstvo azot-monoksida na kortikalnu ekspresiju glukokortikoidnog receptora nije istraženo. Cilj ovog rada bio je da se ispita uticaj endogenog azot-monoksida na strukturu i funkciju kore nadbubrežne žlezde pacova i adrenokortikalnu ekspresiju glukokortikoidnog receptora. Metode. Odrasle ženke pacova Wistar soja u metestrusnoj fazi ciklusa tretirane su Nω-nitro-L-arginin metil esterom (L-NAME), inhibitorom sve tri izoforme azotmonoksid sintaze, u dozi 30 mg/kg, subkutano. Koncentracije adrenokortikotropnog hormona (ACTH) i kortikosterona u krvi određene su radioimunološkom metodom. Na parafinskim presecima nadbubrežne žlezde izvršena je stereološka i imunohistohemijska analiza korteksa. Rezultati. Blokada sinteze azot-monoksida značajno povećava koncentracije ACTH i kortikosterona u krvi. Stereološka analiza je pokazala da tretman sa L-NAME značajno smanjuje numeričku gustinu ćelija u svim kortikalnim zonama. U skladu sa smanjenom numeričkom gustinom, L-NAME značajno povećava volumen ćelija u sve tri zone korteksa. Inhibicija sinteze azot-monoksida smanjuje ekspresiju glukokortikoidnog receptora u zoni fascikulati i zoni retikularis. Zaključak. Dobijeni rezultati ukazuju na mogućnost da endogeni azot-monoksid inhibiše aktivnost kore nadbubrežne žlezde i da moduliše ekspresiju glukokortikoidnog receptora.

Background: This study was performed to investigate expression and distribution of glucocorticoid receptor (GR) in the rat adrenal cortex, acute effect of ethanol on its expression and possible role of endogenous nitric oxide (NO) in this phenomenon. Methods: Adult female Wistar rats showing diestrus day 1 were treated with: a) ethanol (2 or 4 g/kg body weight (b.w.), ip), b) N-omega-nitro-L-arginine methyl ester (L-NAME), well-known competitive inhibitor of all isoforms of NO synthase (NOS), (30 mg/kg b.w., sc) followed by ethanol (4 g/kg, ip) 3 h later and c) L-NAME (30 mg/kg b.w., sc) followed by saline (ip) 3 h later. Untreated rats were used as controls. Adrenocortical expression of GR was estimated by immunohistochemistry. Results: Strong nuclear GR staining was observed throughout the cortex of control rats. Acute ethanol treatment significantly decreased the expression of GR in the zona fasciculata and zona reticularis. Blockade of NO formation had no influence on this effect of ethanol, whereas L-NAME itself induced significant decline in GR immunoreactivity. Conclusions: Obtained findings are the first to demonstrate localization and distribution of the GR throughout the rat adrenal cortex and to suggest that ethanol as well as endogenous NO may modulate adrenocortical expression of this steroid receptor.

Purpose: We compared the gene expression profile of peripheral blood CD34(+) cells and granulocytes in subjects with chronic myeloid leukemia (CML), with the accent on signaling pathways affected by BCR-ABL oncogene. Methods: The microarray analyses have been performed in circulating CD34(+) cells and granulocytes from peripheral blood of 7 subjects with CML and 7 healthy donors. All studied BCR-ABL positive CML patients were in chronic phase, with a mean value of 2012 +/- SD of CD34(+) cells/mu l in peripheral blood. Results: The gene expression profile was more prominent in CML CD34(+) cells (3553 genes) compared to granulocytes (2701 genes). The 41 and 39 genes were significantly upregulated in CML CD34(+) cells (HINT1, TXN, SERBP1) and granulocytes, respectively. BCR-ABL oncogene activated PI3K/AKT and MAPK signaling through significant upregulation of PTPN11, CDK4/6, and MYC and reduction of E2F1, KRAS, and NFKBIA gene expression in CD34(+) cells. Among genes linked to the inhibition of cellular proliferation by BCR-ABL inhibitor Imatinib, the FOS and STAT1 demonstrated significantly decreased expression in CML. Conclusion: The presence of BCR-ABL fusion gene doubled the expression quantity of genes involved in the regulation of cell cycle, proliferation and apoptosis of CD34(+) cells. These results determined the modified genes in PI3K/AKT and MAPK signaling of CML subjects.