Az egysejtű Tetrahymena szignalizációs rendszerének vizsgálata = Investigations on the signalling system of the unicellular Tetrahymena

1. PI 3-kináz aktivitásra utaló jeleket kaptunk, ami wortmanninnal gátolható volt. Megállapítottuk a p85 immunreaktivitás lokalizációját és a: kapcsolatot a p85- és a g-tubulin tartalmú struktúrák között. A wortmannin kezelések befolyásolták a sejtek mikrofilamentáris rendszerét. 2. Megfigyeltük, hogy a különböző mikrotubulus- mérgek eltérő módon gátolják a mikrotubuláris struktúrák kialakulását. A hiszton-deacetiláz gátlók befolyásolják a tubulin acetilációt és a szignalizációs foszfolipidek szintézisét. A taxol kezelések ésgátolják a transzverzális mikrotubulusok kialakulását, hatnak a mitokondriumok szerkezetére. 3. A mikrotubulus polimerizációt gátló anyagok Tetrahymenában ellenkező hatást váltottak ki. 4. A digoxin termelődése hormonálisan befolyásolható. 5 Tetrahymenában nemcsak a teljes endokrin rendszer képviselői vannak jelen, hanem hormonális interakciók is. Természetes körülmények között a víz a hormonokat felhígítja, igy az interakcióknak vagy sejten belül kell történnie, vagy a receptornak kell rendkivül érzékenynek lennie. Ezt bizonyítottuk. Az eredmények meg magyarázzák miért vannak jelen a hormonjok egysejtűekben és hogy használja ezeket az egysejtű természetes körülmények között. 6. Éhezéskor a sejtek inzulin tartalma és kötődése nőtt. 7. Alkalmaztunk egy új fixálási módszert, az EDAC-ot. 8. Kimutattuk a T3 és melatonin jelenlétét és a hormon hatását az egysejtűre. 9. Számos molekula kemotaktikus hatásának vizsgálatában vettünk részt.. | 1. There is a PI 3-kinase activity, which can be inhibited by wortmannin. The localization of p85 immunoreactivity was determined as well as the connection between the p85 and g-tubulin containing structures. Wortmannin treatments influenced microfilaments. 2. The different inhibitory effects of microbular poisons were observed.Histone-deacetylase inhibitors influenced tubulin acetylation and the synthesis of signalizing phospholipids.Taxol treatments hindered the formation of transversal microtubules and disturbed mitochondria. 3. Microtubule polimerization inhibitors had opposite effects in Tetrahymena. 4. The hormonal influence of digoxin production was observed. 5. Not only the representatives of the complete endocrine system are present in Tetrahymena, but also hormonal interactions. In natural watery conditions the hormones are extremely dissolved, so the interactions mast happen inside the cell, or the receptors are rather sensitive. This was justified experimentally. The results can explain why the hormones are present in protozoa and how they are used in natural conditions. 6. Starvation elevates the insulin content and binding. 7. New EDAC fixation was used in Tetrahymena. The presence of T3 and melatonin as well as the effect of the latter on Tetrahymena was demonstrated. 9. We participated in the study of chemotaxis of numerous molecules

Funkcionális genomikai rendszer kidolgozása gyermekkori acut lymphoid leukémiában DNS és cDNS chip analízis valamint immunfenotipizálás alapján = Development of a novel functional genomic system in pediatric acute lymphoid leukemia based on DNA and cDNA chip analysis as well as immunophenotyping

Olyan molekuláris markerek genomikai identifikálását tűztük ki célul, melyek a gyermekkori akut limfoid leukémia (ALL) patomechanizmusának felderítésében, osztályzásának pontosításában és a kezelésre adott válasz predikciójában szerepet játszhatnak. A blaszt-minták gyűjtésének akadályozottsága miatt projektünkben a génpolimorfizmusok vizsgálata kapott fő hangsúlyt. Létrehoztunk egy nemzetközi viszonylatban is kimagaslóan nagynak számító biobankot ALL-es és osteosarcomás gyermekek továbbá egészséges véradók genomi DNS-mintáiból. Az ABCB1 1236C/T, 2677G/T,A és 3435T/C, ABCC1 2012G/T, ABCC2 1249G/A és 3563T/A, ABCC3 -211C/T, ABCG2 34G/A és 421C/A, SLC19A1 80G/A és GGH -401C/T génpolimorfizmusok meghatározását végeztük, és korreláltattuk a betegek adataival. Eredményeink szerint az ABCB1 és ABCG2 vizsgált polimorfizmusai nem hajlamosítanak ALL kialakulására. Megállapítottuk, hogy az ABCB1 3435T/C genotípus prediktív értékkel bír a vizsgált kemoterápiás protokoll kapcsán kialakuló immunszuppresszió vonatkozásában. Az ABCB1 3435TT genotípus és az ABCG2 421A allél hordozása szinergisztikus interakció révén hajlamosít a citosztatikus szerek központi idegrendszeri mellékhatásaira ALL esetében. A GGH 401TT genotípusú betegek metotrexát-eliminációja gyorsabbnak bizonyult mind az ALL-es, mind az osteosarcomás betegek adagolási sémája esetén, és súlyos szövődmények is ritkábban lépnek fel. Ezen eltérések alapját képezhetik a terápia-individualizációt előkészítő további tanulmányoknak | Our objective was to identify molecular markers in the genome that play important role in the pathomechanism, accurate classification and response to therapy in childhood acute lymphoblastic leukemia (ALL). Given the fact that the collection of malignant blast samples was highly hampered, we mainly concentrated on studying somatic gene polymorphisms. A large DNA-biobank was established consisting of samples from pediatric ALL, osteosarcoma and healthy blood donors. The ABCB1 1236C/T, 2677G/T,A and 3435T/C, ABCC1 2012G/T, ABCC2 1249G/A and 3563T/A, ABCC3 -211C/T, ABCG2 34G/A and 421C/A, SLC19A1 80G/A, GGH -401C/T polymorphisms were genotyped and the association of genotype and clinical data were examined. In our study, the polymorphisms of ABCB1 and ABCG2 were not related to susceptibility to ALL. We found that the ABCB1 3435T/C genotype is predictive for the severe immunosuppression developed during the given ALL chemotherapy protocol. The ABCB1 3435TT genotype and carrying ABCG2 421A allele exert synergistic interaction in predisposing patients to the central nervous system adverse effects of cytostatic drugs in ALL. The elimination of methotrexate proved to be faster in patients of GGH -401TT genotype in case of both chemotherapy regimens used in ALL and osteosarcoma, and severe side effects of methotrexate was also more rare in this group. These results may lay the foundation of further studies to develop individual drug dosing protocols

Hormone (ACTH, T3) content of immunophenotyped lymphocyte subpopulations

Cells of the immune system synthesize, store, and secrete polypeptide and amino acid type hormones, which also influence their functions, having receptors for different hormones. In the present experiment immunophenotyped immune cells isolated from bone marrow, thymus, and peritoneal fluid of mice were used for demonstrating the adrenocorticotropic hormone (ACTH) and triiodothyronine (T3) hormone production of differentiating immune cells. Both hormones were found in each cell type, and in each maturation state, which means that all cells are participating in the hormonal function of the immune system. The lineage-independent presence of ACTH and T3 in differentiating hematopoietic cells denotes that their expression ubiquitous during lymphocyte development. Higher ACTH and T3 content of B cells shows that these cells are the most hormonally active and suggests that the hormones may have an autocrine regulatory role in B cell development. Developing T cells showed heterogeneous hormone production which was associated with their maturation state. Differences in the hormone contents of immune cells isolated from different organs indicate that their hormone production is defined by their differentiation or maturation state, however, possibly also by the local microenvironment

Természetes ölő (NK) T lymphocyták légúti gyulladásokban = Natural killer (NK) T lymphocytes in airway inflammation

1.A természetes ölősejtek által keltett légúti gyulladás szteroid gyulladásgátlóval szemben rezisztens. 2.Ebben a modellben csökken az allergiával szembeni tolerancia és fokozódik egy viszonylag újabban felfedezett gyulladáskeltő mediátor, az interleukin-17 (IL-17) szintézise a tüdőben. 3. Az IL-17 hiánya a fenti modellben szteroid rezisztenssé teszi a légúti hyperreaktivitást. 4. Szteroid rezisztens légúti allergia (asthma) modellünkben microarray technikával találtunk mintegy 23 olyan gént, melyeknek a szteroid rezisztencia kialakulásában szerepe lehet. 5. Terhes asthmás betegekben igazoltuk, hogy a Treg sejtek számbeli fokozódása elmarad, mely egészséges terhességben egyébként jellemző. Az anyai asthma tehát csökkenti a terhesség alatti immuntoleranciát. 6. Ugyanerre utalt a szérum HSP70 koncentráció, valamint a Th17/Treg sejtarány emelkedése is asthmás terhesekben. 7. Egészséges terhesekben összefügg a Treg sejtek számának fokozódása (immuntolerancia) a magzati növekedés fiziológiás mértékével. Asthmás terhesekben a Treg szám nem nő és nincs összefüggés a Treg szám és a magzati születési súly között. 8.Számos eredmény született a dohányzás által kiváltott kísérletes pulmonalis hypertonia pathomechanizmusáról, a cisplatinnal kapcsolatos nephropathia kockázati télnyezőiről, valamint az asthma és a tüdőrák különféle biomarkereiről. | 1.Airway inflammation induced by the activation of natural killers proved to be resistant to treatment by steroid antiinflammatory agents. 2.In this model, tolerance against the development of allergies is decreased and the synthesis of interleukin-17 (IL-17), a recently discovered proinflammatory mediator, is increased. 3.Absence of IL-17 in this model of asthma makes airway inflammation steroid resistant. 4.In our earlier, steroid resistant airway allergy model, using microarray technique, we have identified 23 genes possibly responsible for steroid resistance. 5.The increase of Treg cells in healthy pregnancy is absent in pregnant patients suffering from asthma. Thus, asthma interferes with natural immunotolerance of pregnancy. 6.The same conclusion can be drawn basen on the increase of heat-shock protein-70 and the Th17/Treg ratio in asthmatic pregnants. 7.There is a relationship between the abundance of maternal Tregs and the birth weight of newborns in healthy pregnants. This relationship is absent in asthmatic pregnants as is the abundance of Tregs. 8. Many new findings have been published by us about the mechanism of experimental pulmonary hypertension induced by smoking and nephropathy induced by cisplatin (in lung cancer patients). New biomarkers (in the exhaled breath) were identified in asthmatics and lung cancer patients

Both Freshly Prepared and Frozen-Stored Amniotic Membrane Cells Express the Complement Inhibitor CD59

Amniotic membrane proved to be very effective tool in the treatment of a number of ocular surface diseases. The amniotic membrane, however, has to be stored before its transplantation onto the ocular surface followed by mandatory serologic control in order to exclude the transmission of certain viruses. Therefore it is most important to study if cryopreservation of the membrane affects cell surface expression of the molecules. We measured cell surface expression of CD59, a membrane-bound complement inhibitor on the cells of freshly prepared and cryopreserved amniotic membrane. Cells of amniotic membrane were separated mechanically. Epithelial and mesenchymal cells were identified by the intracellular expression of nanog and the cell surface ICAM1 positivity, respectively. Multicolor flow cytometric immunophenotyping was used for determination of the CD59 expression. CellQuest-Pro software program (Becton Dickinson) was used both for measurements and analysis. CD59-positive cells could be detected in all investigated samples and in all investigated cell types, although the expression level of CD59 differed. CD59 was expressed both on freshly prepared and frozen-stored samples. Higher level of CD59 was detected on ICAM1+ mesenchymal cells than on nanog+ epithelial cells. Our findings indicate that amniotic membranes maintain their complement inhibiting capacity after cryopreservation

Neutrophil-to-Lymphocyte Ratio Is an Independent Risk Factor for Coronary Artery Disease in Central Obesity

Several inflammatory biomarkers were found to be associated with an increased risk of cardiovascular disease. Neutrophil-to-lymphocyte ratio (NLR) is a marker of subclinical inflammation that increases with the stress response. Visceral adiposity index (VAI) calculated as a combination of anthropometric and metabolic parameters reflects both the extent and function of visceral adipose tissue. Given the association of subclinical inflammation with both obesity and cardiovascular diseases, it is plausible that the inflammation-CVD association is modulated by the amount and function of adipose tissue. Thus, our aim was to examine the association between NLR and coronary artery calcium score (CACS), an intermediate marker of coronary artery disease in asymptomatic patients across VAI tertiles. Methods: Data from 280 asymptomatic participants of a cardiovascular screening program were analysed. In addition to the collection of lifestyle and medical history, a non-contrast cardiac CT scan and laboratory tests were performed on all participants. Multivariate logistic regression was conducted with CACS > 100 as the outcome and with conventional cardiovascular risk factors and NLR, VAI, and NLR by VAI tertile as predictors. Results: We found an interaction between VAI tertiles and NLR; NLR values were similar in the lower VAI tertiles, while they were higher in the CACS > 100 in the 3rd VAI tertile (CACS ≤ 100: 1.94 ± 0.58 vs. CACS > 100: 2.48 ± 1.1, p = 0.008). According to multivariable logistic regression, the interaction between NLR and VAI tertiles remained: NLR was associated with CACS > 100 in the 3rd VAI tertile (OR = 1.67, 95% CI 1.06-2.62, p = 0.03) but not in the lower tertiles even after adjustment for age, sex, smoking, history of hypertension, hyperlipidaemia, and diabetes mellitus, as well as high-sensitivity C-reactive protein. Our findings draw attention to the independent association between subclinical, chronic, systemic inflammation and subclinical coronary disease in obesity

A sejtek közti kommunikáció újonnan azonosított mikrovezikulum-útjának vizsgálata = Analysis of cell-derived microvesicles that represent novel players in intercellular communication

Munkánk során az extracelluláris vezikulák izolálásának és detektálásának számos meghatározó preanalitikai és analitikai paraméterére hívtuk fel a figyelmet. Elsőként mutattunk rá, hogy a mikrovezikulák és a fehérje-aggregátumok biofizikai paraméterei jelentős mértékben átfednek, és ez zavarhatja a mikrovezikulák mérését. Kidolgoztuk annak módszerét, hogy egyazon biológiai forrásból származó különböző vezikula populációkat párhuzamosan, nagy mennyiségben, intakt formában tudjunk izolálni. Összehasonlító proteomikai elemzést végeztünk thymus eredetű apoptotikus testek és mikrovezikulák esetében. Számos T sejt jelátvitelben és immunfolyamatokban szerepet játszó fehérjét és autoantigént azonosítottunk. Igazoltuk, hogy T sejt eredetű citokinek és extracelluláris vezikulák együttes hatását monociták génexpressziójára. Igazoltuk, hogy a mikrovezikulák önálló ionháztartással rendelkeznek. Kimutattuk, hogy thymocyta exoszómák nem tartalmaznak riboszómális RNS-eket, azonban feldúsulnak bennük kis RNS-ek (pl. bizonyos miRNS-ek). Polymyositises betegekben emelkedett keringő mikrovezikula számot mutattunk ki, mely korrelált a betegség bizonyos klinikai paramétereivel. Végül elsőként igazoltuk, hogy egészséges T sejt eredetű mikrovezikulák CD62P-CD161 kölcsönhatás révén specifikusan kötődnek monociták felszínéhez. | In our work we drove attention to several pre-analytical and analytical parameters affecting isolation and detection of work extracellular vesicles. We were the first to describe that microvesicles share biophysical parameters with protein aggregates which may confound microvesicle assessment by flow cytometry. We developed protocols for the isolation of large amounts of intact vesicle types secreted simultaneously by the same biological source. We carried comparative proteomic analysis of murine thymus derived apoptotic bodies and microvesicles. We identified large number of proteins involved in T cell signaling or immune functions as well as autoantigens within these structures. We provided evidence fro crosstalk between T cell derived extracellular vesicles and cytokines on the gene expression of monocytes. We have shown that microvesicles possess autonomous ion homeostasis. We found that thymocyte derived exosomes lacked the 18S and 28S ribosomal RNA molecules, while they were enriched in small RNA species (e.g. certain miRNAs). We described that patients with polyomyelitis were characterized by elevated levels of circulating microvesicle. Monocyte- and B cell-derived microvesicle numbers correlated with certain clinical parameters of the diseases. Finally, for the first time we showed that HLA-G+, trophoblast-derived microvesicles isolated from healthy pregnant blood plasma samples, bound specifically to T cells via CD62P-CD161 interaction, and induced STAT3 phosphorylation

Unravelling the Role of Trophoblastic-Derived Extracellular Vesicles in Regulatory T Cell Differentiation

Regulatory T cells (Treg) are mandatory elements in the maintenance of human pregnancy, but their de novo differentiation has not been completely exposed. HSPE1 chaperone expressing trophoblast cells may have a role in it. Trophoblast-derived extracellular vesicles (EVs), either at the feto–maternal interface or in circulation, target CD4+ T cells. We hypothesized that HSPE1-associated trophoblastic cell line (BeWo)-derived EVs are active mediators of Treg cell differentiation. We proved at first that recombinant HSPE1 promote human Treg cell differentiation in vitro. Developing a CRISPR-Cas9 based HSPE1 knockout BeWo cell line we could also demonstrate, that EV-associated HSPE1 induces Treg development. Next-generation sequencing of miRNA cargo of BeWo-EVs characterized the regulatory processes of Treg polarization. By the use of single-cell transcriptomics analysis, seven Treg cell subtypes were distinguished and we demonstrated for the first time that the expression level of HSPE1 was Treg subtype dependent, and CAPG expression is characteristic to memory phenotype of T cells. Our data indicate that HSPE1 and CAPG may be used as markers for identification of Treg subtypes. Our results suggest, that trophoblastic-derived iEVs-associated HSPE1 and miRNA cargo have an important role in Treg cell expansion in vitro and HSPE1 is a useful marker of Treg subtype characterization

Monocyte activation drives preservation of membrane thiols by promoting release of oxidised membrane moieties via extracellular vesicles

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.The redox state of cellular exofacial molecules is reflected by the amount of available thiols. Furthermore, surface thiols can be considered as indicators of immune cell activation. One group of thiol containing proteins, peroxiredoxins, in particular, have been associated with inflammation. In this study, we assessed surface thiols of the U937 and Thp1 monocyte cell lines and primary monocytes in vitro upon inflammatory stimulation by irreversibly labelling the cells with a fluorescent derivative of maleimide. We also investigated exofacial thiols on circulating blood mononuclear cells in patients with rheumatoid arthritis and healthy controls. When analysing extracellular vesicles, we combined thiol labelling with the use of antibodies to specific CD markers to exclude extracellular vesicle mimicking signals from thiol containing protein aggregates. Furthermore, differential detergent lysis was applied to confirm the vesicular nature of the detected extracellular events in blood plasma. We found an increase in exofacial thiols on monocytes upon in vitro stimulation by LPS or TNF, both in primary monocytes and monocytic cell lines (p<0.0005). At the same time, newly released extracellular vesicles showed a decrease in their exofacial thiols compared with those from unstimulated cells (p<0.05). We also found a significant elevation of surface thiols on circulating monocytes in rheumatoid arthritis patients (p<0.05) and newly released extracellular vesicles of isolated CD14(+) cells from rheumatoid arthritis patients had decreased thiol levels compared with healthy subjects (p<0.01). Exofacial peroxiredoxin 1 was demonstrated on the surface of primary and cultured monocytes, and the number of peroxiredoxin 1 positive extracellular vesicles was increased in rheumatoid arthritis blood plasma (p<0.05). Furthermore, an overoxidised form of peroxiredoxin was detected in extracellular vesicle-enriched preparations from blood plasma. Our data show that cell surface thiols play a protective role and reflect oxidative stress resistance state in activated immune cells. Furthermore, they support a role of extracellular vesicles in the redox regulation of human monocytes, possibly representing an antioxidant mechanism.This work was supported by the National Scientific Research Program of Hungary (OTKA) grant no. PD 104369 to KSZT, grant no. PD 112085 to VVK and grant no. 111958 and 120237 to EIB, the MEDINPROT Program (Synergy programs I, III and IV), BMBS COST Action(BM1202), the János Bolyai Research Fellowship of the Hungarian Academy of Sciences (to KVV) and the Kerpel Fronius Program of the Semmelweis University (Astellas Pharma Grant to BSW)