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

Matične ćelije opredeljene za granulocitno-monocitnu lozu kostne srži i periferne krvi svinja

The pig is widely used as a large animal model for biomedical research and could be an interesting experimental model for studies of the hematopoietic system and its response in physiological and pathological conditions. With the intention of using the pig as a large animal model in hematopoietic research, a clonal assay in methylcellulose was developed and standardized for detection of committed progenitors of the granulocyte-macrophage lineage from adult pig bone marrow and peripheral blood. Progenitor cells were stimulated to proliferate and differentiate in vitro by adding pig leukocyte conditioned medium (LCM) as a source of homologous growth factors. The number of CFU-GM (Colony Forming Unit -Granulocyte-Macrophage) directly depended on the concentration of LCM. The proliferative rate of CFU-GM progenitor cells was determined by the cytosine arabinoside suicide technique. The percentage of bone marrow and peripheral blood CFU-GM cells in S phase of the cell cycle was 34.7% and 22.2%, respectively. The data obtained regarding the number and characteristics of pig bone marrow and peripheral blood CFU-GM confirmed that the organization of the pig CFU-GM progenitor cell compartment is similar and comparable to that in miniature swine, other animal species and humans.Svinja je životinja koja se koristi kao model u različitim biomedicinskim istraživanjima, a mogia bi biti i interesantan model u ispitivanju fiziologije i patolofizioloških promena hematopoetskog sistema. U cilju razvoja eksperimentalnog modela svinje u istraživanju hematopoeze, stanadardizovan je esej za odredivanje i karakterizaciju opredeljenih matičnih ćelija granulocitno-monocitne loze iz kostne srži i krvi odrasle svinje. Stimulacija proliferacije i diferencijacije ovih matičnih ćelija postignuta je dodavanjem medijuma kondicioniranog leukocitima (LCM - Leukocyte conditioned medium) bogatog faktorima rasta. Broj CFU-GM (Colony forming unit- granulocyte-macrophage) je direktno zavisio od koncentracije LCM-a. Procenat CFU-GM ćelija u S fazi ćelijskog cikiusa odredjivan je tehnikom 'suicida' korišćenjem citozin arabinozida (Ara-C) i iznosio je 34.7% za CFU-GM iz kostne srži i 22.2% za CFU-GM iz periferne krvi. Podaci dobijeni za broj i karakteristike CFU-GM iz kostne srži i periferne krvi potvrđuju da je ovaj odeljak matičnih ćelija kod odraslih svinja organizovan na isti način kao i kod minijaturnih svinja, drugih vrsta životinja i ljudi

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

Over-expressed CmbT multidrug resistance transporter improves the fitness of Lactococcus lactis

U ovom radu je izučavan uticaj povećane ekspresije cmbT gena, odgovornog za sintezu CmbT MDR transportera, na rast Lactococcus lactis. L. lactis pripada grupi bakterija mlečne kiseline (BMK) i ima veliku primenu u prehrambenoj industriji kao starter kultura. CmbT transporter je nedavno okarakterisan MDR protein soja L. lactis, koji doprinosi rezistenciji na različite toksične agense kao i na neke klinički značajne antibiotike. U ovom radu je cmbT gen višestruko eksprimiran u soju L. lactis NZ9000 dodavanjem nizina kao inducera. Povećana ekspresija cmbT gena je praćena metodom reverzne transkripcije (RT-PCR). Pokazano je da se nakon dodatka subinhibitornih koncentracija nizina u medijum za rast povećava količina sintetisane informacione RNK specifične za cmbT gen. Rast soja L. lactis NZ9000/pCT50, u kome je višestruko eksprimiran cmbT gen i L. lactis NZ9000 kontrolnog soja praćen je u bogatom i hemijski definisanom medijumu u prisustvu samo metionina (0.084 mM) ili kombinacije metionina i cisteina (8.4 mM i 8.2 mM). Praćene su krive rasta oba soja, a nakon izračunavanja odgovarajućih vremena generacije, rezultati su pokazali da L. lactis NZ9000/pCT50, brže raste u odnosu na kontrolni soj. Uočena razlika je najverovatnije posledica aktivnosti CmbT transportera koji doprinosi izbacivanju toksičnih agenasa iz ćelije i na taj način poboljšava adaptivne sposobnosti bakterije koja ga eksprimira i daje joj selektivnu prednost.The influence of the over-expression of CmbT multidrug resistance transporter on the growth rate of Lactococcus lactis NZ9000 was studied. L. lactis is a lactic acid bacteria (LAB) widely used as a starter culture in dairy industry. Recently characterized CmbT MDR transporter in L. lactis confers resistance to a wide variety of toxic compounds as well as to some clinically relevant antibiotics. In this study, the cmbT gene was over-expressed in the strain L. lactis NZ9000 in the presence of nisin inducer. Over-expression of the cmbT gene in L. lactis NZ9000 was followed by RT-PCR. The obtained results showed that the cmbT gene was successfully over-expressed by addition of sub-inhibitory amounts of nisin. Growth curves of L. lactis NZ9000/pCT50 over-expressing the cmbT gene and L. lactis NZ9000 control strain were followed in the rich medium as well as in the chemically defined medium in the presence solely of methionine (0.084 mM) or mix of methionine and cysteine (8.4 mM and 8.2 mM, respectively). Resulting doubling times revealed that L. lactis NZ9000/pCT50 had higher growth rate comparing to the control strain. This could be a consequence of the CmbT efflux activity, which improves the fitness of the host bacterium through the elimination of toxic compounds from the cell

Hipogalaktozilacija imunoglobulina G (IgG) gingivalne tečnosti i pljuvačke u obolelih od parodontopatije

Changed glycosylation of immunoglobulin G (IgG), above all, the expression of thermal galactose, influence to numerous functions of those immunoglobulin and correlate with the inflammatory level in a number of diseases. Aim: This work analyses the distribution of IgG subclasses and the content of thermal galactose in them, in saliva and gingival fluid of the patients with periodontal disease and different gum inflammatory level. Materials and methods: It was used saliva and gingival fluid of 30 adults with clinical picture of periodontal disease and 20 persons with healthy periodontium. The qualification of IgG was done by “dot-blot” procedure and the, and thermal galactose was determined by lectin immunoblot procedure. Results: The division of IgG subclasses in both fluids was different in the patients with periodontal disease and in control samples. In saliva and gingival fluid of the diseased quantitatively dominated IgG2 subclasses, independently from periodontal status. In IgG of both fluids, thermal galactose was exprimated at the healthy periodontium persons (control) and with the person with initial periodontal disease, while at the person with increased periodontal disease the expression of this saccharide wasn’t registered in neither of fluids. Conclusion: The results showed that there is a shift towards hypogalactosyled IgG glikoforms during the process of gum inflammation at the periodontal disease patients.Promenjena glikozilacija imunoglobulina G (IgG), pre svega ekspresija terminalne galaktoze, utiče na brojne funkcije ovih imunoglobulina i korelira sa stepenom zapaljenja u mnogim bolestima. Cilj rada: U ovom radu analizirana je distribucija IgG podklasa i sadržaj terminalne galaktoze u njima, u pljuvačci i gingivalnoj tečnosti bolesnika sa parodontopatijom različitog stepena inflamacije gingive. Materijal i metod: Kao materijal u ispitivanjima korišćena je gingivalna tečnost i pljuvačka 30 odraslih osoba sa kliničkom dijagnozom parodontopatija i 20 osoba sa zdravim parodoncijumom. Kvantifikacija IgG urađena je “dot-blot” postupkom, a određivanje terminalne galaktoze lektinskim imunoblot postupkom. Rezultati: Raspodela IgG podklasa u obe tečnosti se razlikovala kod parodontopatija i u kontrolnim uzorcima. I u pljuvačci i u gingivalnoj tečnosti obolelih, kvantitativno je dominirala IgG2 podklasa, nezavisno od parodontalnog statusa. U IgG obe oralne tečnosti terminalna galaktoza je bila eksprimirana kod osoba sa zdravim parodoncijumom (kontrola) i kod osoba sa početnom (inicijalnom) parodontopatijom, dok kod osoba sa uznapredovalom parodontopatijom ekspresija ovog šećera nije registrovana ni u jednoj od ove dve tečnosti. Zaključak: Rezultati ovih istraživanja ukazuju da postoji pomeranje prema hipogalaktozilovanim IgG glikoformama tokom procesa inflamacije gingive u obolelih od parodontopatije

Virulence potential of multidrug-resistant Acinetobacter baumannii isolates from COVID-19 patients on mechanical ventilation: The first report from Serbia

Since the WHO declared the COVID-19 pandemic in March 2020, the disease has spread rapidly leading to overload of the health system and many of the patients infected with SARS-CoV-2 needed to be admitted to the intensive care unit (ICU). Around 10% of patients with the severe manifestation of COVID-19 need noninvasive or invasive mechanical ventilation, which represent a risk factor for Acinetobacter baumannii superinfection. The 64 A. baumannii isolates were recovered from COVID-19 patients admitted to ICU at General Hospital “Dr Laza K. Lazarević” Šabac, Serbia, during the period from December 2020 to February 2021. All patients required mechanical ventilation and mortality rate was 100%. The goal of this study was to evaluate antibiotic resistance profiles and virulence potential of A. baumannii isolates recovered from patients with severe form of COVID-19 who had a need for mechanical ventilation. All tested A. baumannii isolates (n = 64) were sensitive to colistin, while resistant to meropenem, imipenem, gentamicin, tobramycin, and levofloxacin according to the broth microdilution method and MDR phenotype was confirmed. In all tested isolates, representatives of international clone 2 (IC2) classified by multiplex PCR for clonal lineage identification, blaAmpC, blaOXA-51, and blaOXA-23 genes were present, as well as ISAba1 insertion sequence upstream of blaOXA-23. Clonal distribution of one dominant strain was found, but individual strains showed phenotypic differences in the level of antibiotic resistance, biofilm formation, and binding to mucin and motility. According to PFGE, four isolates were sequenced and antibiotic resistance genes as well as virulence factors genes were analyzed in these genomes. The results of this study represent the first report on virulence potential of MDR A. baumannii from hospital in Serbia

Proliferation and differentiation markers of colorectal adenocarcinoma and their correlation with clinicopathological factors

Background/aim: The purpose of this study was to investigate proliferation and differentiation markers in colorectal adenocarcinoma and their correlation with clinicopathological factors. Materials and methods: Samples were collected from 38 patients with colorectal adenocarcinoma and 10 healthy controls. E-cadherin, carcinoembryonic antigen (mCEA), cyclin B1, vascular endothelial growth factor (VEGF), and erythropoietin (EPO) receptor (EPOR) were examined by immunohistochemistry; VEGF and EPO were examined by real-time PCR. Results: The tumor samples were mostly characterized by large dimension (pT3), moderate level of differentiation (G2), negative lymph node status (N0), and no metastasis. Cyclin B1 and VEGF gene and protein expressions were significantly higher in tumor tissues than in control tissues; E-cadherin expression was significantly decreased in tumor samples and in positive correlation with mCEA. EPO was almost undetectable in tumor tissues of colorectal adenocarcinoma. Significant positive correlation was detected between tumor size and cyclin B1, tumor grade, and lymph node status. Conclusion: Decreased expression of EPO, high levels of VEGF and cyclin B1 expression, predominant moderate tumor differentiation, absence of metastasis, and negative lymph node status may suggest low level of aggressiveness, better prognosis, and longer patient survival

Mesenchymal stem cell properties of dental pulp cells from deciduous teeth

In the present study we have isolated and identified mesenchymal stem cells (MSCs) from the exfoliated deciduous teeth dental pulp (DP-MSCs), as plastic-adherent, spindle-shaped cells with a high proliferative potential. Immunophenotype analyses revealed that DP-MSCs were positive for mesenchymal cell markers (CD90, CD44, CD105, STRO-1, vimentin and α-SMA), and negative for hematopoietic stem cell markers (CD11b, CD33, CD34, CD45, CD235a). DPMSCs were also capable of differentiating into adipogenic, chondrogenic, myogenic and osteogenic lineages, fulfilling the functional criterion for their characterization. These results demonstrate that DP-MSCs offer a valuable, readily accessible source to obtain and store adult stem cells for future use

Odnos između totalnog kapaciteta za vezivanje gvožđa i koncentracije transferina kod novorođene prasadi tretirane gvožđe-dekstranom

Serum iron concentration and iron saturation of transferrin (Trf) are measures of body iron stores after administration of iron supplements. In clinical and experimental research, the complex determination of Trf was replaced by the simple determination of total iron binding capacity (TIBC). The objective of this work was to define if TIBC could be an adequate measure for Trf in neonatal piglets after i.m. iron administration. Treated piglets received 150 mg of iron-dextran i.m. the first day of life, and were compared to the untreated control group. Prior to iron administration, as well as on days 2, 8 and 12 after iron administration, serum iron and TIBC concentration were analyzed by an automatized chemical analyzer and Trf was determined by densitometry of electrophoretic strips. Our results show that regardless of iron treatment, TIBC is not a measure of Trf concentration in neonatal piglets two days after birth. At day 8 of their life a high correlation coefficient of these two parameters was established in non-treated animals, while in iron-treated piglets the same correlation was established 12 days after iron treatment. Thus, we suggest that in neonatal piglets, TIBC could be used as a measure of Trf concentration only 12 days after i.m. iron treatment.Određivanje statusa gvožđa u organizmu jedinke posle primene određenog preparata ovog mikroelementa moguće je utvrditi određivanjem njegove koncentracije u serumu i zasićenja transferina (Trf) gvožđem. U kliničkoj i eksperimentalnoj praksi složeno određivanje koncentracije Trf zamenjeno je jednostavnim određ ivanjem ukupnog serumskog kapaciteta za vezivanje gvožđa (TIBC). Cilj ovog rada je bio da se na modelu porasta serumskog Fe po i.m. aplikaciji Fe-dextrana novorođenoj prasadi, utvrdi odnos TIBC i Trf po aplikaciji ovog mikroelementa, kako bi se utvrdilo da li visoke doze gvožđa u serumu utiču na vrednost TIBC kao mere za određivanje koncentracije Trf. Vrednosti za serumsko Fe, TIBC i Trf poređene su između grupe životinja koja je odmah po rođenju dobila 150 mg Fe-dextrana i kontrolne grupe u kojoj životoinje nisu tretirane Fe-dextranom. Krv je uzorkovana pre aplikacije Fe-dextrana, drugog, osmog i dvanaestog dana po aplikaciji preparata gvožđa. Koncentracija gvožđa u serumu i TIBC su određivani standarnim kliničkim biohemijskim analizama, dok je koncenracija Trf određena denzitometrijom elektroforetskih traka. Dobijeni rezultati ukazuju da bez obzira na primenu preparata Fe, TIBC nije adekvatna mera za Trf kod novorođene prasadi u prva tri dana po rođenju. Osmog dana života prasadi, utvrđen je visoki stepen korelacije ova dva parametra kod životinja koje nisu bile tretirane, dok je kod tretiranih jedinki taj stepen korelacije postignut dvanaestog dana. Na osnovu izloženih rezultata se može zaključiti da se kod novorođene prasadi TIBC može koristiti kao mera za Trf tek 12 dana nakon i.m. tretmana Fe-dextranom