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The titanium implant was treated with plasma electrolytic oxidation and subsequent ionic exchange and thermal treatment in order to obtain bioactive layer consisting of titanium oxide, calcium and sodium titanates and hydroxyapatite, as confirmed by X-ray diffraction (XRD). Scanning electron microscopy (SEM) revealed that the given method, besides corresponding phase composition, enables suitable nanotopology for cell attachment and proliferation. Cytotoxicity investigations by MTT, LDH and propidium iodide assays and light microscopy showed that these coatings were not toxic to L929 cells.

The aim of this study was to investigate the biocompatibility of nanostructured materials based on highly active calcium silicates mixed with different radiocontrast agents in comparison to MTA(+) using in vitro and in vivo model. Morphology of materials' samples was analyzed using SEM while the phase compositions were identified by XRD. pH values of materials' suspensions were conducted by pH-meter. The cytotoxicity of materials' solutions was tested by MTT test (100, 50, 25 and 12.5mg/ml). LDH and H-3-thymidine assay were utilized for biocompatibility investigations of materials' eluates (24h, 7 day and 21 day). Eighteen Guinea pigs were used for intramuscular implantation, as teflon tubes with freshly prepared materials were placed into intramuscular pockets. All samples were composed of round and needle-like particles equally distributed with Ca/Si ratio 2.7 at%, with the presence of hydrated calcium silicate phases. The pH values of ALBO-MPCA(1) and ALBO-MPCA(2) were high alkaline, while in case of MTA(+) they were lower and continuously declined (p lt 0.05). Investigated materials didn't exhibit dose-dependent effect on metabolic activity of L929 cells (p>0.05). Significant differences in the percentage of cytotoxicity between diluted and undiluted extracts between all tested materials after 24h and 7 day were noticed (p lt 0.05). Increase in L929 cells proliferation was noticed in case of undiluted eluates of ALBO-MPCA(1) and ALBO-MPCA(2) after 7 day (p lt 0.05). There were no statistically significant differences in the intensity of inflammatory response between investigated materials and control group after 60 day (p>0.05). Evaluation of biocompatibility of both ALBO-MPCA(1) and ALBO-MPCA(2) indicate their potential clinical use. [GRAPHICS] .

Background/Aim: Lately, fully innovative sol-gel method with high-temperature self-propagating reaction was used for the synthesis of new nanostructured endodontic materials, in combination with different radiopacifiers: bismuth (ALBO-MPCA1) and barium (ALBO-MPCA2). The aim of this study was to investigate the biocompatibility of nanostructured endodontic materials based on highly active calcium silicates and mixed with different radiopacifiers in comparison to MTA+ using human stem cells from the apical papilla- SCAP cells. Material and Methods: Morphology of the samples was studied by SEM. The tested materials were mixed with distilled water in a ratio 2:1 (m/m). Fifteen minutes after the preparation, samples were used in the experiment. The biocompatibility of fresh materials, after 3h and 7 days, was tested using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide- MTT test. Results: Samples mostly consisted of spherical and rode-like. The relative viability of cells increased following the exposure time. Conclusion: The biocompatibility of synthesized materials is comparable to the control material MTA+, and therefore these materials can be recommended for further clinical studies.

Kunić, kao eksperimentalna životinja, je veoma čest model u raznovrsnim biomedicinskim istraživanjima, zahvaljujući sličnosti u morfološkim i kliničkim osobinama sa ljudskim, kao i lakoći odgajivanja, držanja i posmatranja. Za izbor modela u bimedicinskim istraživanjima, neophodno je dobro poznavati anatomsku građu, morfološke i fiziološke karakteristike eksperimentalnih životinja. Kunić, kao životinja sa indukovanom ovulacijom, je model kod koga se precizno može utvrditi vreme ovulacije, a po sličnosti fetalnog i embrionalnog razvića sa ljudskim, predstavlja model, koji u ispitivanjima iz oblasti reprodukcije ima prednost nad drugim eksperimentalnim životinjama. Uzimajući u obzir da vrsta kunića, pol, starost, kao i način držanja utiču na neke morfološke i fiziološke karakteristike ove vrste, ova ispitivanja izvedena su na Kalifornijskom kuniću, sa ciljem da se utvrde i upotpune podaci o topografiji ženskih polnih organa Kalifornijskog kunića (Oryctolagus cuniculus domesticus), njihovoj vaskularizaciji, histološkim promenama u ženskom reproduktivnom traktu kunića, tokom različitih faza polnog ciklusa, kao i njegovoj imunološkoj reaktivnosti tokom ovih faza. Ispitivanja su vršena na 30 polno zrelih ženki, prosečne starosti 5-7 meseci, telesne mase oko 3200-4000 g, koje su parene prvi put, ili su bile u graviditetu po prvi put. Anatomskim metodama rada izvršena su topografska ispitivanja ženskih reproduktivnih organa kunića, pelvimetrija i vaskularizacija, za koju je korišćena i rendgenografija. Isečci tkiva jajnika, jajovoda, materice, rodnice i usmine bojeni su hematoksilin eozinom, Gomori metodom i metodom po Gordon-Sweet-u, za histološka ispitivanja. Kriostatski isečci materice i vagine bojeni su metodom peroksidaznog obelezavanja ćelija, radi ispitivanja imunoloških karakteristika ovih organa. Po svojim topografskim i morfološkim osobinama, reproduktivni ograni ženki kunića su slični reproduktivnim organima ženki karnivora i činčile. A. ovarica i a. uterina su g, Rabbit, as an experimental model at various biomedical investigations, thanks to its similarity in many morphological and clinical performances with humans, associated with its breeding facilities, carriage and observation. Knowledge of anatomic, morphologic and physiologic characteristics of experimental animal is necessary for the model selection at biomedical researches. Rabbit, as an induced ovulator, is model with precisely defined time of ovulation; its similarities in fetal and embryo development with humans, give an advantage to rabbit as reproductive model. Considering that breed, gender, age, different way of housing, affect some morphological and physiological performance at certain breed, those investigation was carried out on Californian rabbit (Oryctolagus cuniculus domesticus), with an aim to determine and fulfill the data about topography of rabbit female reproductive tract, vascularization, histological changes during the different phases of reproductive cycle and immunological reactivity during the cycle. The investigation was taken at 30 sexually mature females, age about 5-7 months, body weight about 3200-4000 g, mated for the first time, or at first pregnancy. Standard anatomical methods were used for study in topography, pelvimetry and vascularization that is examined also with rendgenography. Tissue samples of ovarium, Falopian tube, uterus and vagina were stained with haematoksylin eosin, Gomori’s method and Gordon-Sweet’s method, for histological examination. Cryosections of different anatomical parts of uterus and vagina, were stained by peroxidase labbeling the cells, to evaluate the immunological status of those tissues. Similirities with females of Carnivora and Chinchilla, in topography and morphology of reproductive tract were found. The principal blood vessels of rabbit female genital tract are a. ovarica and a. uterina. Histological and immunological changes during the different phases of reproductive cycle were evident. T-cells and

Варфарин (4-хидроксикумарин) је антагонист витамина К. Овај кумарински дериват се користи као антикоагулантни родентицид и као терапеутско средство у профилакси тромбоемболијских болести. Антикоагулантни ефекат варфарина се заснива на инхибицији корака, који зависе од витамина К, у синтези бројних фактора коагулације у јетри. Захваљујући инхибицији циклуса витамина К, варфарин утиче и на друге протеине неопходне за биолошке процесе ван хемостазе (раст и калцификацију костију, раст глаткомишићних ћелија васкулатуре и мезангијских ћелија и друге), доводећи до штетних последица. Варфарин инхибира и процесе који нису у вези са витамином К, укључујући раст тумора. Малобројни подаци указују да овај агенс може да делује и на поједине аспекте имунске функције. Механизми деловања варфарина на имунски систем су, међутим, најмање познати. Ова дисертација је имала за циљ да испита имуномодулаторни потенцијал варфарина након његове епикутане и оралне примене код пацова. Акутни епикутани третман (три дана за редом у дози од 10 μg и 100 μg натријум варфарина) одговара професионалној или акциденталној изложености, а субакутни орални третман (30 дана у дози од 0.35 mg/L и 3.5 mg/L натријум варфарина у пијаћој води) одговара терапијској примени. Проинфламаторни и имуномодулаторни потенцијал варфарина је испитан анализом хуморалних параметара (концентрација акутно фазних протеина и проинфламаторног цитокина, интерлеукина 6 (IL-6) у плазми, као и активности основних ензима антиоксидативне одбране супероксид дисмутазе и каталазе у еритроцитима), као и ћелијских параметара [квантитативне и квалитативне промене леукоцита периферне крви, посебно полиморфонуклеарних леукоцита (PMN)] као показатеља запаљења на системском нивоу. Механизми дејства на полиморфонуклеарне ћелије периферне крви испитани су у погледу њихове оксидативне активности, продукције проинфламаторних цитокина, фактора некрозе тумора (TNF) и IL-6, способности адхезије, миграције и екстравазације у циљна ткива, коришћењем мод, Warfarin (4-hydroxycoumarin) is a vitamin K antagonist. This coumarin derivative is used as an anticoagulant rodenticide and as a therapeutic agent for the prophylaxis of thromboembolic disorders. The anticoagulant effect of warfarin is based on the inhibition of the steps which are dependent on Vitamin K in the synthesis of a number of coagulation factors in the liver. Owing to the inhibition of the vitamin K cycle, warfarin also affects the other proteins which are necessary for the biological processes apart from hemostasis (growth and calcification of bones, vascular smooth muscle cells, mesangial cells and others), leading to harmful effects. Warfarin also inhibits the processes which are not related to vitamin K, including the growth of tumors. Few data suggest that this agent may act on certain aspects of immune function. Mechanisms of action of warfarin on the immune system are, however, the least known. This study was aimed to investigate the immunomodulatory potential of warfarin after its epicutaneous and oral administration in rats. The acute epicutaneous treatment (within three consecutive days in doses of 10 μg and 100 μg sodium warfarin) corresponds to professional or accidental exposure, and the subacute oral treatment (30 days in doses of 0.35 mg/L and 3.5 mg/L sodium warfarin in drinking water) corresponds to the therapeutic use. The proinflammatory and immunomodulatory potential of warfarin has been tested by analyzing the humoral parameters (concentration of acute phase proteins and pro-inflammatory cytokines, interleukin 6 (IL-6) in the plasma, as well as the activities of basic enzymes of antioxidant defence, superoxide dismutase and catalase in erythrocytes) as well as the cell parameters [quantitative and qualitative changes in peripheral blood leukocytes, especially polymorphonuclear leukocytes (PMN)] as an indicator of inflammation at the system level. Mechanisms of effects to the peripheral blood polymorphonuclear cells were tested with re

Prolaktin (PRL) je polipeptidni hormon koji utiče na rast i diferencijaciju različitih ćelija, a uključen je i u brojne fiziološke procese. Sintetiše ga i luči hipofiza, ali i različita druga tkiva i ćelije. Prolaktin je jedan od glavnih proteina koji sintetiše i sekretuje decidualizovani endometrijum. Produkcija decidualnog prolaktina se povećava u trudnoći nakon implantacije, i dostiže maksimum u tkivu decidue 20 do 25 nedelje gestacije. Delovanje PRL se ostvaruje vezivanjem za PRL receptore (PRLR) na ćelijskoj membrani. Poznato je da se za prolaktinski receptor vezuje najmanje tri liganda - prolaktin, placentni laktogen i hormon rasta. Do sada je za placentu poznato da je receptor za prolaktin ispoljen u decidui, trofoblastu horiona, epitelu amniona i sinciciotrofoblastu na kraju trudnoće. Prolaktin u različitim ćelijama i tkivima utiče na ekspresiju adhezionih i proteolitičkih molekula koji su značajni za degradaciju ekstraćelijskog matriksa i ćelijsku migraciju. Međutim, njegova uloga u trofoblastu nije dovoljno poznata. Ovim radom je po prvi put ispitivan uticaj prolaktina na svojstva invazivnog ekstravilusnog trofoblasta prvog trimestra trudnoće. Detektovana je ekspresija PRLR, kao i profil prisutnih formi receptora na izolovanim citotrofoblastnim ćelijama i HTR-8/SVneo trofoblastnoj imortalizovanoj ćelijskoj liniji. Pokazali smo da PRL stimuliše adheziju, migraciju i invaziju trofoblasta in vitro. Kao potencijalne efektore ispitivali smo integrine, gal-1 i matriksne metaloproteinaze-2 i -9. Dobijeni rezultati ukazuju da PRL stimuliše ekspresiju subjedinica integrina α1, α5, kao i gal-1. Ispitivali smo i efekat PRL na vijabilnost, proliferaciju i apoptozu HTR-8/SVneo ćelija. PRL je blago stimulisao vijabilnost i uticao na povećanje broja adherentnih ćelija, dok apoptoza nije bila značajno promenjena. Dobijeni rezultati predstavljaju napredak u razumevanju fiziološke uloge PRL značajne za funkciju humanog trofoblasta. Takođe, ovaj rad pruža celovitiji uvid u p, Prolactin (PRL) is a polypeptide hormone which has impact on the growth and differentiation of various cell types, and is known to participate in numerous physiological processes. It is synthesized and secreted by the pituitary gland and many other tissues and cell types. Prolactin is also one of the major proteins synthesized and secreted by decidualized endometrium. The production of decidual prolactin increases after implantation, and peaks in decidual tissue at 20 to 25th week of gestation. The action of PRL is exerted through binding to its receptors (PRLR) at the surface of target cells. It is known that prolactin receptor has at least three ligands – prolactin, placental lactogen and growth hormone. Regarding localization of prolactin receptor in placenta, so far it has been found in decidua, chorionic trophoblast, amniotic epithelium and synciciotrophoblast at term. Prolactin affects the expression of adhesion and proteolytic molecules which are important for degradation of extracellular matrix and cell migration. However, its role in trophoblast is not well defined. In this study the effect of prolactin on the function of first trimester of pregnancy extravillous trophoblast was studied for the first time. The expression of PRLR, as well as the profile of different isoforms was examined in both cytotrophoblast and HTR-8/SVneo trophoblast derived cell line. It is shown here that PRL stimulates trophoblast cell adhesion, migration and invasion in vitro. Potential effectors were sought among integrins, gal-1 and matrix metalloproteinases-2 and -9. The results showed that PRL stimulated the expression of integrin subunits α1, α5, as well as gal-1. When investigating the effect of PRL on cell viability, proliferation and apoptosis of HTR-8/SVneo cells, PRL was found to slightly stimulate cell viability and adherent cell number, while apoptosis was not altered. The results of this study represent a step further in the understanding of the physiological role of PR

Background/Aim. Morbidity and mortality of continous ambulatory peritoneal dialysis (CAPD) patients is still very high. The aim of the study was to evaluate the effects of peritoneal dialysis (PD) solutions (standard vs biocompatible) on long-term patients' and the techique survival. Methods. A total of 42 stable patients on CAPD participated in this crosssectional study. They were prospectively followed-up during the twelve years. Patients with severe anemia (Hb lt 10 g/L) and malignant disease ware excluded. Twenty one (50%) patients were treated with the standard PD solutions (CAPDP- 1) while the other 21 (50%) were treated with biocompatible PD solutions [(lower level of glucose degradation products, lower concentration of Ca2+ and neutral pH (CAPDP-2)]. All patients were analyzed for a presence of vascular calcification, nutrition status, and parameters of inflammation after 2.5 ± 0.6 years of starting CAPD, and these variables considered in the analysis as risk factors. Results. The patients from the group CAPDP-2 compared to those from the group CAPDP-1 had lower level of high-sensitivity C-reactive protein (hs-CRP) (p = 0.003), and better nutritional status as confirmed by the mid-arm circumference (p = 0.015), and midarm muscle circumference (p = 0.002) and subjective global assessment (p = 0.000). Also, they had lower vascular calcifications as confirmed by intima media thickness (IMT) (p = 0.003), degree of carotid narrowing (p = 0.001) and calcified plaques of common carotid arteries (CCA) (p = 0.008). Kaplan- Meier analysis confirmed better survival of patients from the group CAPDP-2 than those from the group CAPDP-1 (1-, 5-, and 10-year patients survival rate was: 100%, 61.9% and 14.3% for the group CAPDP-1, and 100%, 85.7%, and 52.4% for the group CAPDP-2, respectively; p = 0.0345). The 1-, 5-, and 10-year technique survival rate was: 100%, 71.4%, and 38.1% for the group CAPDP-1, and 100%, 85.7%, and 76.2% for the group CAPDP-2, respectively; (p = 0.0, Uvod/Cilj. Morbiditet i mortalitet bolesnika na kontinuiranoj ambulantnoj peritoneumskoj dijalizi (KAPD) i dalje je neprihvatljivo visok. Cilj rada bio je da se proceni uticaj vrste dijaliznih rasvora (bioinkompatibilni vs biokompatibilni) na višegodišnje preživljavanje bolesnika i same tehnike KAPD. Metode. Ovom studijom preseka sa delimično prospektivnim praćenjem ishoda lečenja obuhvaćeno je ukupno 42 nasumice izabrana, stabilna bolesnika (26 muškaraca i 16 žena) lečena primenom metode KAPD tokom poslednjih 12 godina. Isključeni su bolesnici sa teškom anemijom (Hb lt 10 g/L) i malignom bolešću. Pri tome, 21 (50%) bolesnika kontinuirano je lečeno bioinkompatibilnim rastvorom za KAPD (kiseli standardni rastvor - ANDY-disc; grupa KAPDB-1), dok je preostalih 21 bolesnik sve vreme bilo na biokompatibilnijem rastvoru za KAPD (neutralni rastvor sa znatno manjom koncentracijom degradacionih produkata glukoze, 1.25 mmol/L Ca i 40 mmol/L laktata - Gambrosol Trio; grupa KAPDB-2). Svim bolesnicima određeni su odabrani parametri hronične inflamacije, malnutricije i ateroskleroze zajedno sa transportnim karakteristikama peritoneumske membrane i rezidualnom bubrežnom funkcijom nakon 2,5 ± 0,6 god od započinjanja KAPD. Svi dobijeni rezultati analizirani su kao potencijalni faktori rizika. Rezultati. Grupa KAPDB-2 u odnosu na KAPDB-1 imala je statistički značajno niže vrednosti serumskog hs-CRP (p = 0,003) i bolje parametre nutritivnog statusa izražene kroz obim nadlaktice (p = 0,015), obim mišića nadlaktice (p = 0,002) i subjektivnu globalnu procenu (p = 0,000) kao i u manjoj meri prisutnu aterosklerozu potvrđeno debljinom intimomedijalnog kompleksa (IMT) (p = 0,003), stepenom suženja karotida (p = 0,001) i prisustvom kalcifikovanih ateromatoznih plakova na karotidnim arterijama (p = 0,008). Kaplan-Meier-ova kriva preživljavanja potvrdila je značajno duže preživljavanje bolesnika u grupi KAPD-2 u odnosu na KAPDB-1 (1-, 5-, i 10-godišnje preživljavanje bolesnika iznosilo je redom

Background: Chronic inflammation, malnutrition and atherosclerosis (MIA syndrome) are important predictors of high mortality in continuous ambulatory peritoneal dialysis (CAPD) patients. We aimed to evaluate the effects of PD solutions (standard vs. biocompatible) on some parameters of MIA syndrome in patients undergoing CAPD. Methods: 42 stable patients who were on CAPD at least 2.5 years participated in this cross-sectional study. Patients who had severe anemia (Hb lt 10 g/l), immunomodulatory therapy, peritonitis or any inflammatory conditions for at least 3 months before the analysis, malignant disease and acute exacerbation of heart failure, were excluded. 21 (50%) patients were treated with standard PD solutions (CAPDP-1), while the remaining 21(50% of patients) were treated with biocompatible PD solutions (neutral solutions with lower level of glucose degradation products and lower concentration of calcium, CAPDP-2). All patients underwent echocardiography and B-mode ultrasonography of common carotid arteries together with assessments of nutrition status and parameters of systemic and local inflammation. Results: There were no significant differences between the groups concerning age, gender, underlying disease, residual renal function, peritoneal transport characteristics, comorbidity or therapy applied. Patients from group CAPDP-2 had a significantly lower serum level of hs-CRP (3.7 +/- 2.6 mg/l vs. 6.3 +/- 4.5 mg/l; p = 0.023) and significantly better nutritional status confirmed by mid-arm circumference (p = 0.015), mid-arm muscle circumference (p = 0.002) and subjective global assessment (14.28% of patients in CAPDP-2 vs. 71% of patients in CAPDP-1 were malnourished; p = 0.000). Group CAPD-2 had less frequent left ventricular hypertrophy (p = 0.039), thinner intima-media thickness (p = 0.005), smaller carotid narrowing (p = 0.000) and fewer calcified plaques of common carotide arteries (p = 0.003). No significant difference between the CAPDP groups was o

Aim To analyse phenotypic characteristics of antigen-presenting cells (APC), isolated from human periapical lesions by flow cytometry and immunocytochemistry. Methodology Sixteen periapical lesions were digested for 15 min with 0.05% collagenase. Mononuclear cells, separated from other inflammatory cells by density centrifugation, were processed for flow cytometry and/or immunocytochemistry. Single and double immunostainings were performed using monoclonal antibodies specific for human CD45, CD3, CD19, CD14, HLA-DR, CD1a, CD83 and CD123. Results Antigen-presenting cells (HLA-DR+ cells) represented 32.9 +/- 17.8% of total mononuclear cells. Amongst them, B cells (HLA-DR+ CD19(+)) were the predominant APC population, followed by activated macrophages (HLA-DR+ CD14(+)), dendritic cells (DC) (HLA-DR+ CD14(-) CD19(-) CD3(-)) and activated T cells (HLA-DR+ CD3(+)). Based on the predominance of T cells (CD3(+)) or B cells and plasma cells (CD19(+) and CD19(lo), respectively) amongst mononuclear cell infiltrates, lesions were divided into T- and B-types. The percentage of DC in T-type lesions (27.1 +/- 6.8% of total HLA-DR+ cells) was higher, compared with B-type lesions (10.3 +/- 5.2%) (P lt 0.01). Within the DC population, the percentages of CD1a (Langerhans cell type) and CD123 (probably plasmacytoid DC type) did not differ significantly between the groups (P > 0.05). However, the percentage of mature DC (CD83(+)) was significantly higher in T-type periapical lesions (P lt 0.05). Conclusion Flow cytometry and immunocytochemistry are suitable methods for phenotypic analysis of APC after their isolation from human periapical lesions. APC, that were phenotypically heterogeneous, constituted a significant component of infiltrating cells. Lesions with the predominance of T cells were characterized by a higher proportion of mature DC (HLA-DR(+)CD83(+) cells) than lesions with predominance of B cells/plasma cells.

Background. Dendritic cells (DC) have been used for immunotherapy of malignant tumors, different kinds of infections, and other clinical conditions. For that purpose, optimal conditions for the generation of functionally mature DC in vitro are required. Two different protocols for the induction of maturation of monocyte-derived DC (MDDC) were compared in this study. Methods. MDDC were generated in vitro by cultivating adherent monocytes of healthy volunteers with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) during 6-days period. The immature DC thus prepared were induced to mature using two protocols. DC were stimulated for 2 days with lipopolysaccharide (LPS), or with a cocktail of proinflammatory mediators (PM) containing IL-1b, IL-6, tumor necrosis factor α (TNFα), and prostaglandin E2 (PGE2), respectively. Phenotypic characteristics of MDDC and their endocytic activity were studied by flow cytometry. Allostimulatory activity of these cells was tested in the mixed leukocyte reaction (MLR), whereas the production of cytokines was determined by ELISA kits. Results. MDDC matured with PM (PM-DC) were predominantly non-adherent cells, while about 30% of LPS-matured DC were adherent cells. In comparison with LPS-DC, PM-DC expressed higher levels of CD86 and CD83, had lower endocytic activity, produced higher levels of IL-10 and lower levels of IL-12, and more strongly stimulated proliferation of allogeneic lymphocytes. Conclusion The protocol based on the combination of proinflammatory cytokines and PGE2 is better for the induction of maturation of human MDDC in vitro than the protocol using LPS alone., Uvod. Dendritične ćelije (DC) se koriste u imunoterapiji malignih tumora, različitih vrsta infekcija i drugih oboljenja. U tom cilju neophodni su optimalni uslovi za dobijanje funkcionalno zrelih DC in vitro. U ovom radu smo poredili dva različita protokola za indukciju maturacije DC monocitnog porekla (MDDC). Metode. MDDC su dobijene in vitro kultivisanjem adherentnih monocita zdravih dobrovoljnih davalaca krvi pomoću faktora stimulacije granulocitno-makrofagnih kolonija (GM-CSF) i interleukina-4 (IL-4) u toku 6 dana. Tako pripremljene nezrele DC su indukovane na sazrevanje pomoću dva protokola. DC su stimulisane u toku 2 dana lipopolisaharidom (LPS) ili koktelom proinflamatornih medijatora (PM) koji je sadržavao IL-1b, IL-6, faktor nekroze tumora α (TNFα) i prostaglandin E2 (PGE2). Fenotipske karakteristike MDDC i njihova endocitozna aktivnost su ispitivani pomoću protočne citometrije. Alostimulatorna aktivnost ovih ćelija je ispitivana u testu mešane leukocitne reakcije (MLR), dok je stvaranje citokina određivano ELISA kompletima. Rezultati. MDDC koje su sazrevale u prisustvu PM (PM-DC) su bile predominantno neadherentne ćelije, dok su oko 30% DC koje su sazrevale pod uticajem LPS (LPS-DC) bile adherentne. U poređenju sa LPS-DC, PM-DC su ispoljavale više nivoe CD86 i CD83 molekula, imale slabiju endocitoznu aktivnost, produkovale više IL-10, a manje IL-12 i snažnije stimulisale proliferaciju alogenih limfocita. Zaključak. Protokol koji se bazira na primeni kombinacije proinflamatornih citokina i PGE2 je bolji za indukciju maturacije humanih MDDC in vitro nego LPS protokol.

In our previous work we showed that 3F10 monoclonal antibody (mAb), which recognizes the rat complement receptor 1-related/gene protein y (Crry), induces homotipic aggregation of thymocytes. In this work we studied the effect of 3F10 mAb on proliferation of rat thymocytes stimulated with concanavalin A (ConA) or by cross-linking the T cell receptor (TCR) by anti-alphabetaTCR mAb (R73), in vitro, and the mechanisms involved in the process. Our results show that 3F10 mAb stimulates proliferation of total thymocytes triggered by suboptimal concentrations of ConA or TCR cross-linking, in a dose-dependent manner. Maximal stimulation was observed using 10 mug/ml and 20 mug/Ml of 3F10 mAb, respectively. The 3F10-induced stimulation of thymocytes proliferation in the presence of ConA, that was followed by increased production of interleukin-2 (IL-2), up-regulation of the expression of IL-2 receptor alpha (IL-2Ralpha) and was inhibited by anti-CD11a and anti-CD 18 mAbs. Purified thymocytes did not respond by proliferation to 3F10 mAb, either alone or in combination with R73 mAb or ConA. Proliferation of these cells was achieved only in the presence of OX-6(+) antigen-presenting cells (APC) and additional signals transmitted by TCR or ConA. These results suggest that Crry is involved in the LFA-1 dependent proliferation of thymocytes, a phenomenon that has not been recognized so far.

Several laboratories have developed culture systems that allow the generation of large numbers of human dendritic cells (DC) from monocytes using granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-4 (IL-4). In this work we provided evidence that GM-CSF (100 ng/ml) in combination with a low concentration of IL-4 (5 ng/ml) was efficient in the generation of immature, non-adherent, monocyte-derived DC as the same concentration of GM-CSF, and ten times higher concentration of IL-4 (50 ng/ml). This conclusion was based on the similar phenotype profile of DC such as the expression of CD1a, CD80, CD86, and HLA-DR, down-regulation of CD14, and the absence of CD83, as well as on their similar allostimulatory activity for T cells. A higher number of cells remained adherent in cultures with lower concentrations of IL-4 than in cultures with higher concentrations of the cytokine. However, most of these adherent cells down-regulated CD14 and stimulated the proliferation of alloreactive T cells. In contrast adherent cells cultivated with GM-CSF alone were predominantly macrophages as judged by the expression of CD14 and the inefficiency to stimulate alloreactive T cells. DC generated in the presence of lower concentrations of IL-4 had higher proapoptotic potential for the Jurkat cell line than DC differentiated with higher concentrations of IL-4, suggesting their stronger cytotoxic, anti-tumor effect., U više laboratorija su uspostavljeni sistemi za kultivaciju velikog broja humanih dendritičnih ćelija (DĆ) od monocita korišćenjem faktora stimulacije granulocitno--makrofagnih kolonija (GM-CSF) i interleukina-4 (IL-4). U ovom radu je pokazano da je kombinacija GM-CSF (100 ng/ml) i mala koncentracija IL-4 (5 ng/ml) podjednako efikasna za dobijanje nezrelih, neadherentnih, DĆ monocitnog porekla kao i kombinacija GM-CSF sa deset puta većom koncentracijom IL-4 (50 ng/ml). Ovaj zaključak izveden je na osnovu sličnog fenotipskog profila DĆ (ispoljavanje CD1a, CD80, CD86 i HLA-DR, smanjenje ekspresije CD14 i odsustva CD83), kao i slične alostimulatorne aktivnosti ovih ćelija za limfocite T. U kulturama sa nižim koncentracijama IL-4 prisutan je bio veći broj adherentnih ćelija nego u kulturama sa većim koncentracijama IL-4. Međutim, većina ovih ćelija je smanjivala ekspresiju CD14 i stimulisala proliferaciju aloreaktivnih limfocita T. Nasuprot njima adherentne ćelije, diferentovane samo u prisustvu GM-CSF, koje su ispoljavale CD14 i nisu imale sposobnost stimulacije aloreakativnih limfocita T pokazivale su karakteristike makrofaga. DĆ obrazovane u prisustvu manjih koncentracija IL-4 imale su veći potencijal za indukciju apoptoze Jurkat ćelijske linije, a time i snažniji citotoksični, antitumorski efekat nego DĆ diferentovane u prisustvu većih koncentracija IL-4.