Stvaranje azot-monoksida izazvano bradikininom nije dovoljno za indukciju gama-globina

Introduction Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO) production both in primary human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC). Moreover, NO increases γ-globin and fetal hemoglobin levels in human erythroid progenitors. Objective In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase γ-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. Methods The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. Results In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 μM up to 30 minutes). This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS), demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS) mRNA level and eNOS/s-actin ratio in HUVEC (by twofold). In addition, bradykinin failed to increase γ-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce γ/β globin ratio in erythroid progenitors in co-cultures with HUVEC. Conclusion Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce γ-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of γ-globin gene expression.Uvod Hidroksikarbamid, koji se koristi u lečenju hemoglobinopatija, podstiče stvaranje azot-monoksida (NO) kako u primarnim ljudskim endotelnim ćelijama pupčane vene (HUVEC), tako i u izmenjenoj endotelnoj ćelijskoj liniji poreklom iz koštane srži (TrHBMEC). Štaviše, NO povećava stvaranje γ-globina i fetalnog hemoglobina u ljudskim progenitorima eritropoeze. Cilj rada Da bismo ustanovili da li jednostavna fiziološka stimulacija stvaranja NO od komponenti mikrosredine hematopoeze može povećati ekspresiju γ-globinskog gena, ispitivali smo efekte bradikinina, već poznatog stimulatora stvaranja NO. Metode rada Studija je izvedena u zajedničkim kulturama ljudskih progenitora eritropoeze sa TrHBMEC ili HUVEC i ispitivana hemiluminiscentnim merenjem NO posredstvom ozona, kao i primenom kvantitativnog RT-PCR na genskom nivou. Rezultati U skladu s prethodnim izveštajima, pokazali smo da endogeni faktor bradikinin povećava stvaranje NO u endotelnim ćelijama na dozno i vremenski zavisan način (0,1-0,6 μM do 30 minuta). Ovo stvaranje NO u HUVEC i TrHBMEC izazvano bradikininom blokirano je od strane konkurentskih inhibitora NO-sintaze (NOS), pokazujući NOS-zavisnost. Utvrdili smo da bradikinin značajno smanjuje stvaranje iRNK endotelne forme NOS (eNOS), kao i odnos eNOS i β-aktina u HUVEC (dvostruko manje). Pored toga, bradikinin ne povećava ekspresiju iRNK γ-globinskog gena ni u zasebnim progenitorima eritropoeze, niti u zajedničkim kulturama progenitora eritropoeze sa TrHBMEC ili HUVEC posle 24 sata tretmana. Bradikinin ne menja ni odnos γ i β globina u zajedničkim kulturama progenitora eritropoeze sa HUVEC. Zaključak Aktivacija eNO_ izazvana bradikininom dovodi do kratkog i malog povećanja NO u endotelnim ćelijama, što je nedovoljno da podstakne ekspresiju gena za γ-globin. Ovi rezultati naglašavaju važnost povećanog i produženog stvaranja NO radi stimulacije ekspresije γ-globina

Efekti IL-17 na funkcionalnu aktivnost ćelija periferne krvi

Interleukin-17 (IL-17) is a proinflammatory cytokine produced mainly by activated CD4+ and CD8+ T cells, while its specific receptor is ubiquitously distributed. The inflammatory capacity of IL-17 is based on its ability to stimulate a wide range of stromal cells to produce and release a number of proinflammatory mediators, some with a known impact on hematopoiesis particularly granulopoiesis. Recent data indicate a role for IL-17 in the pathogenesis of several inflammatory diseases, transplant rejection and tumor growth. The purpose of this study was to determine functional responses including the respiratory burst, nitric oxide (NO) production, adhesiveness and metabolical activity/viability of human peripheral blood leukocytes (total white blood cells, mononuclear cells and granulocytes) from healthy donors in the presence of recombinant human (rh)IL-17. The obtained results showed that rhIL-17 did not induce significant changes in the respiratory burst, NO production, and metabolical activity of each peripheral blood cell fraction the tested, while a slight increase in phorbol-12-myristate-13-acetate (PMA) stimulated adhesiveness of granulocytes and mononuclear cells was noted. The absence of significant changes in tested functional activities of various peripheral blood cells suggests that IL-17 does not express its proinflammatory ability in steady-state, since the requirement for its action really does not exist.Interleukin 17 (IL-17) je proinflamatorni citokin koga produkuju aktivirane CD4+ i CD8+ T ćelije, dok je njegov receptor ubikvitarno distribuiran. Inflamatorni kapacitet IL-17 se zasniva na njegovoj sposobnosti da stimuliše širok spektar stromalnih ćelija da produkuju i oslobađaju različite proinflamatorne medijatore, među kojima neki imaju efekte na hematopoezu posebno granulopoezu. Dosadašnji podaci ukazuju na ulogu IL-17 u patogenezi različitih inflamatornih bolesti, odbacivanju transplanta i razvoju tumora. Cilj ovog rada je bio da se odrede funkcionalni odgovori, uključujući respiratorni prasak, produkciju azot monoksida (NO), adhezivnost i metaboličku aktivnost/vijabilnost različitih ćelija periferne krvi (ukupnih leukocita, mononuklearnih ćelija i granulocita) zdravih donora, u prisustvu IL-17. Dobijeni rezultati su ukazali da IL-17 ne dovodi do značajnih promena respiratornog praska, produkcije NO i metaboličke aktivnosti ćelija periferne krvi, ali da uzrokuje blago povećanje forbol-12-miristat-13-acetat (PMA) stimulisane adhezivnosti granulocita i mononuklearnih ćelija. Odsustvo značajnih promena u ispitivanim funkcionalnim aktivnostima različitih ćelija periferne krvi, ukazuje da IL-17 ne eksprimira proinflamatorno dejstvo kod zdravih osoba, jer najverovatnije i ne postoji potreba za njegovim delovanjem

Primena neravnotežne plazme u medicini

The potential of plasma applications medicine, the connections to nanotechnologies and the results obtained by our group are reviewed. A special issue in plasma medicine is the development of the plasma sources that would achieve non-equilibrium at atmospheric pressure in an atmospheric gas mixture with no or only marginal heating of the gas, and with desired properties and mechanisms that may be controlled. Our studies have shown that control of radicals or chemically active products of the discharge, such as ROS (reactive oxygen species) and/or NO, may be used to control the growth of the seeds. Simultaneously, a specially designed plasma needle and other sources were shown to be efficient to sterilize not only colonies of bacteria but also plank- tonic samples (microorganisms protected by water) or bio films. Finally, it was shown that a plasma might induce differentiation of stem cells. Non-equilibrium plasmas may be used in detection of different specific markers in medicine. For example proton transfer mass spectroscopy may be employed in the detection of volatile organic compounds without their dissociation and thus as a technique for instantaneous measurement of the presence of markers for numerous diseases.U ovom radu dat je pregled primene plazme u medicini, povezanost sa nanotehnologijama i rezultate na ovom polju koje je postigla naša grupa. Poseban problem u plazma medicini je razvoj izvora plazme koji bi radili u neravnotežnim uslovima na atmosferskom pritisku i u smeši gasova kakva je u atmosferi uz zanemarljivo grejanje gasa i sa željenim karakteristikama koje se mogu podešavati po želji. Naša istraživanja su pokazala da se kontrola prisustva radikala i drugih hemijski aktivnih čestica kao što su reaktivne kiseonične čestice (ROS) i/ili NO, može koristiti za kontrolu klijanja semenki. U isto vreme je dokazano za posebno konstruisanu plazma iglu da može efikasno da steriliše ne samo kolonije bakterija već i planktonske uzorke (mikroorganizme zaštićene vodom) pa i biofilmove. Na kraju, mi smo pokazali da plazma može da indukuje diferencijaciju matičnih ćelija. Neravnotežna plazma se može koristiti za detekciju raznih specifičnih markera u medicini. Na primer masena spektroskopija na bazi izmene protona može da se koristi za detekciju isparivih organskih jedinjenja bez njihove disocijacije i na taj način se može ostvariti trenutna detekcija markera za brojne bolesti iz daha

The effect of a plasma needle on bacteria in planktonic samples and on peripheral blood mesenchymal stem cells

In this paper, we study the application of a plasma needle to induce necrosis in planktonic samples containing a single breed of bacteria. Two different types of bacteria, Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), were covered in this study. In all experiments with bacteria, the samples were liquid suspensions of several different concentrations of bacteria prepared according to the McFarland standard. The second system studied in this paper was human peripheral blood mesenchymal stem cells (hPB-MSC). In the case of hPB-MSC, two sets of experiments were performed: when cells were covered with a certain amount of liquid (indirect) and when the cell sample was in direct contact with the plasma. Most importantly, the study is made with the aim to see the effects when the living cells are in a liquid medium, which normally acts as protection against the many agents that may be released by plasmas. It was found that a good effect may be expected for a wide range of initial cell densities and operating conditions causing destruction of several orders of magnitude even under the protection of a liquid. It was established independently that a temperature increase could not affect the cells under the conditions of our experiment, so the effect could those hPB-MSC that were not protected by a liquid, gas flow proved to produce a considerable effect, presumably due to poor adhesion of the cells, but in a liquid the effect was only due to the plasma. Further optimization of the operation may be attempted, opening up the possibility of localized in vivo sterilization

Kultivacija matičnih i progenitorskih ćelija hematopoeze iz kostne srži hrčka

Hamster, a hibernating animal, is an important experimental model in research on the influence of hypothermia on different physiological processes. A simple procedure for cultivation and identification of hamster hematopoetic stem cells (HSC) and hematopoetic progenitor cells (HPC) is a premise for a successful investigation upon hypothermia effects on hematopoiesis. The aim of this work was to evaluate the utilization of commercially available methylcellulose media (MC) and recombinant mouse and human cytokines for hamster HSC and HPC assays, in order to enable further studies on these cells. Hamster bone marrow mononuclear cells (BMMNC) were plated in MC containing cytokines that support mouse or human HPC growth. Also, BMMNC were resuspended in cytokine supplemented liquid media and incubated for 5 weeks with a four day monitoring of viable cell number. We demonstrated that hamster hematopoietic progenitor cells committed for erythroid lineage and myeloid lineage successfully formed recognizable colonies in both mouse and human MC, while multipotent progenitor cells formed colonies only in mouse MC. We also defined conditions for the evaluation of hamster HSC activity in liquid cultures, based on continuous 5 weeks HSC proliferation. The obtained results verify the utilization of mouse specific MC for further research on hamster HPC biology during hypothermia.Fiziološka hibernacija u koju hrčci ulaze prilikom izlaganja niskim temperaturama, čini ove životinje zanimljivim eksperimentalnim modelom za ispitivanje hematopoeze u uslovima hipotermije. Preduslov za ovo ispitivanje je postojanje jednostavne metode za kultivaciju i identifikaciju hematopoetskih ćelija hrčka. Cilj ovog rada je bio da se ispita mogućnost kultivacije progenitorskih ćelija hematopoeze hrčka u kompletnoj metil celulozi dizajniranoj za kultivaciju mišijih i humanih hematopoetskih ćelija, kao i da se odrede optimalni uslovi za kultivaciju matičnih ćelija hematopoeze hrčka u tečnoj kulturi. Mononuklearne ćelije kostne srži hrčka su posađene u metil celulozu i u tečnu kulturu. Oba medijuma su sadržala kombinacije rekombinantnih mišijih i/ili humanih citokina. Kolonije progenitorskih ćelija opredeljenih za mijelopoezu i opredeljenih za eritropoezu su se formirale u metil celulozi dizajniranoj za kultivaciju mišijih i humanih hematopoetskih ćelija, dok su se primitivnije kolonije sastavljene od oba tipa ćelija (mijeloidna i eritrocitna loza) formirale samo u metil celulozi dizajniranoj za kultivaciju mišijih hematopoetskih ćelija. Osim toga, populacija matičnih ćelija hematopoeze hrčka je proliferisala u tečnim kulturama tokom 5 nedelja bez znakova opadanja proliferativnog potencijala. Ova istraživanja pokazuju da se primenjene metode mogu uspešno koristiti za ispitivanje hematopoeze kod hrčka

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34(+) cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34(+) cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs

Mikrosredina hematopoeze

Steady-state hematopoiesis takes place in the bone marrow permissive microenvironment, composed of stromal cells, as well as extracellular matrix (ECM) components and regulatory molecules, produced by both stromal and hematopoietic cells. Stromal cells are both mezenchymal (endothelial cells fibroblasts, adipocytes, osteoblasts, myocytes, adventitial reticular cells) and hematopoietic (macrophages) in origin, which together provide cell to cell interactions, matrix proteins and growth factors essential for the maintenance, growth and differentiation of hematopoietic stem and progenitor cells. The ECM components (collagen, laminin, fibronectin, thrombospondin proteoglycans, hemonectin and various proteinases participating in their remodeling) are involved in different biological functions such as cell adhesion, binding and presentation of various cytokines and regulation of cell growth. These components serve to localize hematopoietic cells in the specific bone marrow microenvironment and it is suggested that in combination with cytokines are crucial for their compartmentalization. The regulatory molecules of hematopoietic microenvironment are cytokines, which regulate the survival, proliferation and differentiation of hematopoietic cells and cell adhesion molecules, which are responsible for the localization of hematopoiesis to the bone marrow. The activity of cytokines may be greater when the cytokine is present in a membrane-bound or ECM-associated form. Hematopoietic cells could also regulate normal hematopoiesis in an autocrine/paracrine manner, since it was shown that these cells express and secrete various growth factors, cytokines and chemokines.Proces hematopoeze se odvija u definisanoj tkivnoj mikrosredini kostne srži koja ima ključnu ulogu u njegovoj regulaciji. Mikrosredina hematopoeze podrazumeva funkcionalno jedinstvo stromalnih ćelija i ćelijskih produkata (molekuli ekstracelularnog matriksa i regulatorni faktori), koji čine kompleksni molekularni milje u kome se ostvaruju specifične interakcije hematopoetskih ćelija i komponenti mikrosredine. Stromalne ćelije mezenhimalnog porekla (endotelne ćelije, fibroblasti, adipociti osteoblasti, miociti, adventicijske ćelije retikuluma) i nemezenhimalnog porekla (makrofage), deluju na matične ćelije hematopoeze direktno među ćelijskim interakcijama, kao i produkcijom i deponovanjem pojedinih komponenti kompleksnog ekstracelularnog matriksa, a takođe i produkcijom i koncentrovanjem lokalnih citokina i faktora rasta sa hematopoetskim efektima, obezbeđujući na taj način gotovo sve činioce neophodne za proliferaciju i diferencijaciju matičnih ćelija hematopoeze. Komponente ECM (kolagen, laminin, fibronektin, trombospondin, proteoglikani, hemonektin i drugi glikoproteini kao i proteolitički enzimi koji omogućavaju remodelovanje ECM) su uključene u ćelijsku adheziju, vezivanje i prezentaciju različitih citokina i regulaciju ćelijskog rasta. Smatra se da komponente ECM omogućavaju lokalizuju hematopoetskih ćelija u specifičnoj mikrosredini kostne srži, i da u kombinaciji sa citokinima imaju presudnu ulogu u formiranju specifičnih niša. Regulatorni faktori hematopoetske mikrosredine su citokini koji regulišu preživljavanje, proliferaciju i diferencijaciju hematopoetskih ćelija i ćelijski adhezivni molekuli koji su odgovorni za lokalizaciju hematopoeze u kostnoj srži i za posredovanje u fizičkoj vezi između hematopoetskih ćelija i stromalnog tkiva mikrosredine. Ovi molekuli se u mikrosredini kostne srži nalaze kao solubilni, ili vezani za membrane stromalnih ćelija ili za komponente ECM, što može da pojača njihovu aktivnost. Takođe i same hematopoetske ćelije učestviju u regulaciji procesa hematopoeze produkujući citokine koji ispoljavaju autokrino/parakrine efekte

p38 MAPK signaling mediates IL-17-induced nitric oxide synthase expression in bone marrow cells

The effects of interleukin (IL)-17 on nitric oxide (NO) synthase (NOS) expression, as well as the participation of mitogen-activated protein kinases (MAPKs) in IL-17-mediated effects were examined in murine bone marrow cells. The results demonstrated the ability of IL-17 to upregulate the expression of mRNA for both inducible NOS and constitutive, endothelial NOS isoforms, as well as to enhance the phosphorylation of p38 MAPK. Moreover, both the NOS-inducing effect of IL-17 and the in vitro IL-17-mediated inhibition colony forming unit-erythroid (CFU-E) growth were dependent on p38 MAPK activity. The data demonstrating that the in vivo reducing effect of IL-17 on bone marrow CFU-E was prevented by co-treatment with the NOS inhibitor Nw-nitro-l-arginine methyl ester hydrochloride (L-NAME), implied that this effect is mediated through NOS activation. Besides revealing a link between the IL-17, NO, and haematopoiesis, data presented gave an insight into the mechanisms by which IL-17 exerts its modulatory effects on bone marrow cells