Expression and significance of chemokine ligand CCL5 and its receptor CCR5 in glioblastoma

Glioblastom multiforme (GB) je najbolj pogost in najbolj agresiven možganski tumor pri človeku, ki ga opredeljuje visoka stopnja proliferacije in invazije v možgansko tkivo. Zdravljenje je zaradi nepopolne kirurške odstranitve in odpornosti tumorja na radio- in kemoterapijo neuspešno, poleg tega pa je ponovitev GB zelo pogosta. Kemotaktični citokini, oz. kemokini so vpleteni v številne signalne poti, tako v fizioloških pogojih kot tudi v bolezenskih stanjih. Vsebnost CCL5 in CCR5 smo določali na nivoju genskega izražanja z metodo qPCR-RT v GB tkivnih vzorcih bolnikov in primarnih celičnih linijah GB ter na proteinskem nivoju z imunohistokemijsko analizo. CCL5 in CCR5 sta povišano izražena v GB vzorcih bolnikov ter v ponovljenih GB, kar je povezano s slabšo prognozo. Kemokin CCL5 in njegov receptor CCR5 v celicah GB vplivata na njihovo proliferacijo, preživetje in invazijo. Kemotaktično gibanje celic v smeri gradienta CCL5/CCR5 lahko blokiramo z antagonistom receptorja CCR5, Maravirocom (MVR), s katerim smo preverili vpliv inhibicije na preživetje in invazijo celic GB. Z uporabo MTT testa smo pokazali, da MVR nima vpliva na celično preživetje. Z 2D modelom celične invazije pa smo dokazali inhibitorni vpliv MVR na invazijo celic GB, ob prisotnosti signala CCL5.Glioblastoma multiforme (GB) is the most frequent and aggressive brain tumor in human, with high proliferative and diffuse invasive properties. The outcome of the disease is dismal, due to impossible total surgical resection and tumor resistance to irradiation and chemotherapy. Moreover, the tumor often recurs in a period of a few months. Chemotactic cytokines or chemokines are involved in many signaling pathways within physiological homeostasis and in pathologic states. In GB tissues samples from patients and in GB cells we have analyzed CCL5 and CCR5 gene expression using qPCR-RT and CCL5 and CCR5 protein expression using immunohistochemistry. Both CCL5 and CCR5 expressions were increased in GB tissue samples and in recurrent GB tissues, which means very bad prognosis for survival. Chemokine CCL5 and its receptor CCR5 in GB cells are involved in proliferation, cell survival and invasion. We can block CCL5 induced chemotaxis with CCR5 antagonist Maraviroc (MVR). Using this inhibitor, we verified the inhibitory effect on cell survival and invasion of GB cells, when challenged by CCL5. Using MTT assay, we also demonstrated that MVR had no effect on GB cell viability

UPPER SECONDARY SCHOOL STUDENTS\u27 ATTITUDE AND KNOWLEDGE ABOUT VACCINES AND VACCINATION

Svetovni in slovenski problem obotavljanja in necepljenja otrok obstaja že dolgo časa. Na odločitev staršev o cepljenju otrok vpliva zaupanje v zdravstveni sistem in zdravstvene delavce, za ustrezno precepljenost pa je potrebno tudi znanje in pozitivno stališče do cepiv in cepljenja. Slednje se začne razvijati že pri otrocih, zato smo v tej magistrski nalogi želeli preveriti stališča in znanje dijakov gimnazije in srednje strokovne šole ter primerjati podatke glede na program šolanja, spol, letnik in v odvisnosti od tega ali dijaki navajajo, da so bili cepljeni proti otroškim boleznim ali ne. Znanje in stališča dijakov smo preverjali preko spletnega anketiranja 1KA, kjer smo objavili anketni vprašalnik s preizkusom znanja. S programom IBM SPSS smo pridobljene podatke statistično analizirali in pridobili rezultate, da je biološko znanje dijakov pomanjkljivo, saj so na manj kot polovico vprašanj odgovorili pravilno, in da imajo dijaki srednje strokovne šole manj znanja kot dijaki gimnazije. Stališča dijakov so v povprečju pozitivna. Dijaki so neopredeljeni do tematike, kdo bi moral odločati o cepljenju otroka ter farmacevtskih podjetij, ki promovirajo cepljenje. Več znanja in bolj pozitivna stališča glede na spol imajo fantje ter dijaki, ki so bili cepljeni. Najmanj znanja imajo dijaki, ki ne vedo ali so bili v otroštvu cepljeni ali ne.The global and Slovenian problem of hesitation and non-vaccination of children has existed for a long time. Parents’ decision to have their children vaccinated is influenced by trust in the health care system and health care professionals, but adequate vaccination coverage also requires knowledge and a positive attitude toward vaccines and vaccination. Attitudes are influenced at a young age. For this reason, in this master’s thesis, we wanted to examine the attitudes and knowledge of secondary school students. We wanted to compare data according to school programme, gender, grade level and also according to whether students report having been vaccinated against childhood diseases or not. We checked students’ knowledge and attitudes through the 1KA online survey, in which we published a questionnaire with a test. We statistically analysed the obtained data using IBM SPSS. The results of our survey showed that students’ biological knowledge was insufficient. They answered less than half of the questions correctly. In addition, the students of the vocational upper secondary school have less knowledge than the students of the general upper secondary school (grammar school). On average, students\u27 attitudes are positivehowever, their attitude toward who should decide whether to vaccinate a child is unclear, as is their attitude toward pharmaceutical companies that promote vaccination. Regarding gender, males have more knowledge and more positive attitudes, and we observed the same pattern in positive attitudes for students who have been vaccinated. Students who do not know whether or not they were vaccinated in childhood have the least knowledge

Vpliv svinca na privzem mineralov pri sončnici in ječmenu

Namen raziskave je bil ugotoviti, kako prisotnost svinca (10-100 μM) vpliva na prevzem mineralov v poganjke in korenine rastlin. Poleg tega pa so nas zanimale še posledice strupenosti svinca, ki se kažejo na biomasi, višini poganjka, dolžini korenine in ostalih rastnih dejavnikih pri sončnici in ječmenu. Mlade rastline sončnic in ječmena smo gojili v čašah s hidroponiko, ki so vsebovale Hoaglandovo raztopino in različne koncentracije svinca. Opazovali smo prisotnost kloroz in nekroz. Primerjali smo rastne dejavnike ter svežo in suho maso rastlin izpostavljenih svincu in kontrolo brez svinca. Klorofilni status smo določili z analizo vsebnosti pigmentov: klorofila a in b ter karotenoidov. Za analizo privzema mineralov v korenine in poganjke smo uporabili rentgensko fluorescenčno spektroskopijo (XRF), s katero smo določili koncentracije elementov v poganjkih in koreninah. Rezultati so pokazali, da ima Pb večji vpliv na sončnico kot ječmen. Svinec pozitivno vpliva na rast organov večinoma le pri koncentraciji 10 uM, kar je najverjetneje posledica hormeze. Povzroči tudi spremembe vsebnosti klorofilov in karotenoidov. Ob prisotnosti svinca se spremeni vsebnost mineralov v sončnici. Ti minerali so klor, mangan, kalcij in žveplo. Kot posledica delovanja svinca so se na rastlinah pojavile še kloroze in nekroze. Svinec vpliva na privzem mineralov v poganjke in korenine. Prav tako vpliva na višino poganjka in vsebnost klorofilov ter karotenoidov. Povzroča nastanek kloroz in nekroz. Vpliv svinca na sončnico je večji, saj ta spada med akumulacijske rastline in bolj intenzivno sprejema kovine. Ječmen je na kovinske elemente bolj odporen, svinec se v njem težje akumulira in zato povzroči manj sprememb

TRIM28 selective nanobody reduces glioblastoma stem cell invasion

Glioblastoma (GB), is the most common and aggressive malignant primary brain tumour in adults. Intra- and inter-tumour heterogeneity, infiltrative GB cell invasion and presence of therapy-resistant GB stem cells (GSCs) represent major obstacles to favourable prognosis and poor therapy response. Identifying the biomarkers of the most aggressive tumour cells and their more efficient targeting strategies aretherefore, crucial. Recently, transcription factor TRIM28 has been identified as a GB biomarker and, in this study, we have shown high expression of TRIM28 in GB and in low grade gliomas as well as higher expression in GSCs vs. differentiated GB cells, although in both cases not significant. We demonstrated significant in vitro inhibition of GB cells and GSCs invasiveness and spread in zebrafish brains in vivo by anti-TRIM28 selective nanobody NB237. TRIM28 was also enriched in GB (tumour) core and associated with the expression of stem cell genes, but was not prognostic for overall survival. However, based on the above results, we conclude that TRIM28 nanobody NB237 offers a new opportunity as a GB therapeutic tool

CCR5-mediated signaling is involved in invasion of glioblastoma cells in its microenvironment

The chemokine CCL5/RANTES is a versatile inflammatory mediator, which interacts with the receptor CCR5, promoting cancer cell interactions within the tumor microenvironment. Glioblastoma is a highly invasive tumor, in which CCL5 expression correlates with shorter patient survival. Using immunohistochemistry, we identified CCL5 and CCR5 in a series of glioblastoma samples and cells, including glioblastoma stem cells. CCL5 and CCR5 gene expression were significantly higher in a cohort of 38 glioblastoma samples, compared to low-grade glioma and non-cancerous tissues. The in vitro invasion of patients-derived primary glioblastoma cells and glioblastoma stem cells was dependent on CCL5-induced CCR5 signaling and is strongly inhibited by the small molecule CCR5 antagonist maraviroc. Invasion of these cells, which was enhanced when co-cultured with mesenchymal stem cells (MSCs), was inhibited by maraviroc, suggesting that MSCs release CCR5 ligands. In support of this model, we detected CCL5 and CCR5 in MSC monocultures and glioblastoma-associated MSC in tissue sections. We also found CCR5 expressing macrophages were in close proximity to glioblastoma cells. In conclusion, autocrine and paracrine cross-talk in glioblastoma and, in particular, glioblastoma stem cells with its stromal microenvironment, involves CCR5 and CCL5, contributing to glioblastoma invasion, suggesting the CCL5/CCR5 axis as a potential therapeutic target that can be targeted with repositioned drug maraviroc

Anti-Vimentin Nanobody Decreases Glioblastoma Cell Invasion In Vitro and In Vivo

Purpose: Glioblastoma (GBM) is the most common primary brain tumour and one of the deadliest cancers. In addition to late diagnosis and inadequate treatment, the extremely low survival rate is also due to the lack of appropriate therapeutic biomarkers and corresponding therapeutic agents. One of the potential therapeutic biomarkers is the intermediate filament vimentin, which is associated with epithelial-mesenchymal transition (EMT). The purpose of this study was to analyse the effect of the anti-vimentin nanobody Nb79 on cell invasion in vitro and in vivo. To further our understanding of the mechanism of action, we investigated the association between Nb79 and EMT in GBM and GBM stem cells by analysing the expression levels of key EMT-related proteins. Methods: The expression of vimentin in glioma tissues and cells was determined by RT-qPCR. An invasion assay was performed on differentiated glioblastoma cell line U-87 MG and stem cell line NCH421k in vitro as well as in vivo in zebrafish embryos. The effect of Nb79 on expression of EMT biomarkers beta-catenin, vimentin, ZEB-1 and ZO1 was determined by Western blot and immunocytochemistry. Results: Our study shows that vimentin is upregulated in glioblastoma tissue compared to lower grade glioma and non-tumour brain tissue. We demonstrated that treatment with Nb79 reduced glioblastoma cell invasion by up to 64% in vitro and up to 21% in vivo. In addition, we found that the tight junction protein ZO-1 had higher expression on the cell membrane, when treated with inhibitory anti-vimentin Nb79 compared to control. Conclusion: In conclusion, our results suggest that anti-vimentin nanobody Nb79 is a promising tool to target glioblastoma cell invasion