119 results on '"Jeras, Matjaž"'
Search Results
2. Nanofibers with genotyped Bacillus strains exhibiting antibacterial and immunomodulatory activity
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Grilc, Nina Katarina, Zidar, Anže, Kocbek, Petra, Rijavec, Tomaž, Colja, Teja, Lapanje, Aleš, Jeras, Matjaž, Gobec, Martina, Mlinarič-Raščan, Irena, Gašperlin, Mirjana, Kristl, Julijana, and Zupančič, Špela
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- 2023
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3. Mesenchymal Stem Cells in the Musculoskeletal System: From Animal Models to Human Tissue Regeneration?
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Čamernik, Klemen, Barlič, Ariana, Drobnič, Matej, Marc, Janja, Jeras, Matjaž, and Zupan, Janja
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- 2018
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4. Survival of castration-resistant prostate cancer patients treated with dendritic–tumor cell hybridomas is negatively correlated with changes in peripheral blood CD56$^{bright}$CD16$^−$ natural killer cells
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Haque Chowdhury, Helena, Hawlina, Simon, Gabrijel, Mateja, Trkov, Saša, Kreft, Marko, Lenart, Gordan, Cukjati, Marko, Kopitar, Andreja Nataša, Kejžar, Nataša, Ihan, Alojz, Ležaič, Luka, Grmek, Marko, Kmetec, Andrej, Jeras, Matjaž, and Zorec, Robert
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udc:616-006 ,natural killer cells ,rak prostate ,naravne celice ubijalke ,zmanjšanje stopnje resekcije ,prostate cancer ,decreasing resection rates - Published
- 2023
5. Development and bioevaluation of nanofibers with blood-derived growth factors for dermal wound healing
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Bertoncelj, Valentina, Pelipenko, Jan, Kristl, Julijana, Jeras, Matjaž, Cukjati, Marko, and Kocbek, Petra
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- 2014
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6. Development of Nanofibers with Embedded Liposomes Containing an Immunomodulatory Drug Using Green Electrospinning.
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Casula, Luca, Zidar, Anže, Kristl, Julijana, Jeras, Matjaž, Kralj, Slavko, Fadda, Anna Maria, and Zupančič, Špela
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LIPOSOMES ,MONONUCLEAR leukocytes ,NANOFIBERS ,CELL physiology ,ETHYLENE oxide ,ELECTROSPINNING - Abstract
Conventional treatments for chronic wounds are often ineffective, thus new therapeutic approaches are needed, such as the delivery of immunomodulatory drugs that can reduce inflammation, restore immune cell function, and facilitate tissue regeneration. A potential drug for such an approach is simvastatin, which has major drawbacks including poor solubility and chemical instability. With the aim of developing a dressing for wound healing, simvastatin and an antioxidant were incorporated into alginate/poly(ethylene oxide) nanofibers by green electrospinning without the use of organic solvents, thanks to their prior encapsulation into liposomes. The composite liposome–nanofiber formulations exhibited fibrillar morphology (160–312 nm) and unprecedentedly high phospholipid and drug content (76%). Transmission electron microscopy revealed dried liposomes as bright ellipsoidal spots homogeneously distributed over the nanofibers. After nanofiber hydration, the liposomes reconstituted in two size populations (~140 and ~435 nm), as revealed by cutting-edge MADLS
® analysis. Lastly, in vitro assays demonstrated that composite liposome–nanofiber formulations are superior to liposomal formulations due to a better safety profile in keratinocytes and peripheral blood mononuclear cells. Furthermore, both formulations exhibited similarly advantageous immunomodulatory effects, measured as decreased inflammation in vitro. A synergistic combination of the two nanodelivery systems shows promise for the development of efficient dressings for chronic wound treatment. [ABSTRACT FROM AUTHOR]- Published
- 2023
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7. Infuence of MHC on odour perception of 43 chemicals and body odour
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Janeš Damjan, Klun Irena, Vidan-Jeras Blanka, Jeras Matjaž, and Kreft Samo
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major histocompatibility complex (mhc) ,human leukocyte antigens (hla) ,odour perception ,sweat ,methyl hexanoate ,musk ,Biology (General) ,QH301-705.5 - Published
- 2010
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8. In vitro impact of bisphenols BPA, BPF, BPAF and 17β-estradiol (E2) on human monocyte-derived dendritic cell generation, maturation and function
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Švajger, Urban, Dolenc, Marija Sollner, and Jeras, Matjaž
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- 2016
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9. Cultivation of Human Articular Chondrocytes for Autologous Transplantation : Possible Solutions to Problems Arising from Insufficient or Delayed Supply of Autologous Serum
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Malicev, Elvira, Kregar-Velikonja, Nevenka, Krašna, Metka, Bonaca, Orjeta, Wozniak, Gordana, Radosavljevic, Damjan, Jeras, Matjaž, Kneževic, Miomir, Lindner-Olsson, Elisabeth, editor, Chatzissavidou, Nathalie, editor, and Lüllau, Elke, editor
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- 2001
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10. Regulated exocytosis in astrocytic signal integration
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Parpura, Vladimir, Baker, Brandi J., Jeras, Matjaž, and Zorec, Robert
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- 2010
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11. An assay for functional dendritic cell-specific ICAM-3-grabbing nonintegrin (DC–SIGN) inhibitors of human dendritic cell adhesion
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Obermajer, Nataša, Švajger, Urban, Jeras, Matjaž, Sattin, Sara, Bernardi, Anna, and Anderluh, Marko
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- 2010
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12. Homozygous Human TAP Peptide Transporter Mutation in HLA Class I Deficiency
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de la Salle, Henri, Hanau, Daniel, Fricker, Dominique, Urlacher, Arlette, Kelly, Adrian, Salamero, Jean, Powis, Stephen H., Donato, Lionel, Bausinger, Huguette, Laforet, Michel, Jeras, Matjaz, Spehner, Danièle, Bieber, Thomas, Falkenrodt, Annie, Cazenave, Jean-Pierre, Trowsdale, John, and Tongio, Marie-Marthe
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- 1994
13. Late dendritic cells are still able to evoke a potent alloreactive CTL response
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Repnik, Urska, Bergant, Martina, Wraber, Branka, and Jeras, Matjaz
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- 2008
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14. Fused Late Endocytic Compartments and Immunostimulatory Capacity of Dendritic–Tumor Cell Hybridomas
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Gabrijel, Mateja, Bergant, Martina, Kreft, Marko, Jeras, Matjaž, and Zorec, Robert
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- 2009
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15. In Vitro Characterization of the Human Skeletal Stem Cell-like Properties of Primary Bone-Derived Mesenchymal Stem/Stromal Cells in Patients with Late and Early Hip Osteoarthritis.
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Jasenc, Lara, Stražar, Klemen, Mihelič, Anže, Mihalič, Rene, Trebše, Rihard, Haring, Gregor, Jeras, Matjaž, and Zupan, Janja
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HIP osteoarthritis ,STROMAL cells ,GENE expression profiling ,HUMAN stem cells - Abstract
Human skeletal stem cells (hSSCs) were recently identified as podoplanin (PDPN)/CD73/CD164-positive and CD146-negative cells that decline with age, and play a role in the pathogenesis of osteoarthritis (OA). The aim of this study was to identify the hSSC-like properties of bone-derived mesenchymal stem/stromal cells (MSCs) of patients with late and early OA. Methods: First, we performed gene expression profiling for the hSSC markers in 32 patients with late and early OA, and donors without OA. Having identified the low expression of hSSC markers in late OA patients, we further performed trilineage differentiation and immunophenotyping for hSSC makers in the selected subsets from each donor group. Results: Our results show no differences in osteogenesis, chondrogenesis, and adipogenesis between the MSCs from the three groups. However, the immunophenotyping shows lower CD164 in MSCs from early OA patients in comparison with late and no OA subjects (p = 0.002 and p = 0.017). Conclusions: Our study shows that the in vitro hSSC-like properties of bone-derived MSCs are similar in patients with early and late OA, and in donors without OA. However, the lower percentage of CD164-positive MSCs in early OA patients indicates the potential of CD164 as a marker of the onset of OA. [ABSTRACT FROM AUTHOR]
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- 2022
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16. Calcipotriol Affects Keratinocyte Proliferation by Decreasing Expression of Early Growth Response-1 and Polo-like Kinase-2
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Kristl, Jernej, Slanc, Petra, Krašna, Metka, Berlec, Aleš, Jeras, Matjaž, and Štrukelj, Borut
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- 2008
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17. Preparation of native and amplified tumour RNA for dendritic cell transfection and generation of in vitro anti-tumour CTL responses
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Bergant, Martina, Meden, Luka, Repnik, Urška, Sojar, Valentin, Stanisavljević, Dragoje, and Jeras, Matjaž
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- 2006
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18. CNL, a ricin B-like lectin from mushroom Clitocybe nebularis, induces maturation and activation of dendritic cells via the toll-like receptor 4 pathway
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Švajger, Urban, Pohleven, Jure, Kos, Janko, Štrukelj, Borut, and Jeras, Matjaž
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- 2011
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19. Dendritic cells treated with resveratrol during differentiation from monocytes gain substantial tolerogenic properties upon activation
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Švajger, Urban, Obermajer, Nataša, and Jeras, Matjaž
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- 2010
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20. Evaluation of a fibrin-based skin substitute prepared in a defined keratinocyte medium
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Krasna, Metka, Planinsek, Franc, Knezevic, Miomir, Arnez, Zoran M., and Jeras, Matjaz
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- 2005
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21. Niflumic acid renders dendritic cells tolerogenic and up-regulates inhibitory molecules ILT3 and ILT4
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Švajger, Urban, Vidmar, Alenka, and Jeras, Matjaž
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- 2008
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22. Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues
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Kos, Janko, Sekirnik, Andreja, Premzl, Aleš, Bergant, Valentina Zavašnik, Langerholc, Tomaž, Repnik, Urška, Turk, Boris, Werle, Bernd, Golouh, Rastko, Jeras, Matjaž, and Turk, Vito
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- 2005
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23. Survival of castration‐resistant prostate cancer patients treated with dendritic–tumor cell hybridomas is negatively correlated with changes in peripheral blood CD56brightCD16− natural killer cells.
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Haque Chowdhury, Helena, Hawlina, Simon, Gabrijel, Mateja, Trkov Bobnar, Saša, Kreft, Marko, Lenart, Gordan, Cukjati, Marko, Kopitar, Andreja Nataša, Kejžar, Nataša, Ihan, Alojz, Ležaič, Luka, Grmek, Marko, Kmetec, Andrej, Jeras, Matjaž, and Zorec, Robert
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CASTRATION-resistant prostate cancer ,PROSTATE cancer patients ,KILLER cells ,HYBRIDOMAS ,CYTOTOXIC T cells - Abstract
Dear Editor, We investigated the clinical outcome of treating castration-resistant prostate cancer (CRPC) patients with autologous immunohybridoma cell (aHyC) vaccine generated by electrofusing autologous dendritic (DC) and tumor cells (TC), and tested whether the immunological response, involving the CD56 SP bright sp CD16 SP - sp natural killer (NK), putative pro-metastatic cells,1,2 correlates with survival of CRPC patients. For each patient, the data collected from all questionnaires in the period before the first autologous hybridoma cell (aHyC) vaccine application were averaged, and the same was done for the data acquired during/after the treatment (twice per patient). Survival of castration-resistant prostate cancer patients treated with dendritic-tumor cell hybridomas is negatively correlated with changes in peripheral blood CD56
bright CD16- natural killer cells Human NK lymphocytes are involved in antitumor immunity, and CD56 SP bright sp CD16 SP - sp NK cells are considered immunoregulatory cytokine-producing cells, representing 5%-10% of all NK cells in peripheral blood.9 The levels of counterpart CD56 SP dim sp CD16 SP + sp NK cells were unaltered compared to baseline in both groups. [Extracted from the article]- Published
- 2021
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24. Determination of optimal transport conditions for biopsies of human articular cartilage as well as for suspensions of cultured chondrocytes used in autologous transplantation
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Maličev, Elvira, Wozniak, Gordana, Velikonja, Nevenka Kregar, Knežević, Miomir, and Jeras, Matjaž
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- 2001
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25. Vitamin C induced apoptosis in human articular chondrocytes
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Maličev, Elvira, Wozniak, Gordana, Knežević, Miomir, Radosavljević, Damijan, and Jeras, Matjaž
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- 2000
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26. IFN-γ-induced increase in the mobility of MHC class II compartments in astrocytes depends on intermediate filaments
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Vardjan Nina, Gabrijel Mateja, Potokar Maja, Švajger Urban, Kreft Marko, Jeras Matjaž, de Pablo Yolanda, Faiz Maryam, Pekny Milos, and Zorec Robert
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Astrocytes ,Vesicle mobility ,Late endosomes/lysosomes ,Major histocompatibility class II compartments ,Interferon-γ ,Dextran labeling ,Immune response ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Abstract Background In immune-mediated diseases of the central nervous system, astrocytes exposed to interferon-γ (IFN-γ) can express major histocompatibility complex (MHC) class II molecules and antigens on their surface. MHC class II molecules are thought to be delivered to the cell surface by membrane-bound vesicles. However, the characteristics and dynamics of this vesicular traffic are unclear, particularly in reactive astrocytes, which overexpress intermediate filament (IF) proteins that may affect trafficking. The aim of this study was to determine the mobility of MHC class II vesicles in wild-type (WT) astrocytes and in astrocytes devoid of IFs. Methods The identity of MHC class II compartments in WT and IF-deficient astrocytes 48 h after IFN-γ activation was determined immunocytochemically by using confocal microscopy. Time-lapse confocal imaging and Alexa Fluor546-dextran labeling of late endosomes/lysosomes in IFN-γ treated cells was used to characterize the motion of MHC class II vesicles. The mobility of vesicles was analyzed using ParticleTR software. Results Confocal imaging of primary cultures of WT and IF-deficient astrocytes revealed IFN-γ induced MHC class II expression in late endosomes/lysosomes, which were specifically labeled with Alexa Fluor546-conjugated dextran. Live imaging revealed faster movement of dextran-positive vesicles in IFN-γ-treated than in untreated astrocytes. Vesicle mobility was lower in IFN-γ-treated IF-deficient astrocytes than in WT astrocytes. Thus, the IFN-γ-induced increase in the mobility of MHC class II compartments is IF-dependent. Conclusions Since reactivity of astrocytes is a hallmark of many CNS pathologies, it is likely that the up-regulation of IFs under such conditions allows a faster and therefore a more efficient delivery of MHC class II molecules to the cell surface. In vivo, such regulatory mechanisms may enable antigen-presenting reactive astrocytes to respond rapidly and in a controlled manner to CNS inflammation.
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- 2012
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27. POSSIBLE IMPACT OF THE HPA-COMPATIBILITY ON THE OUTCOME OF THE BONE MARROW TRANSPLANTATION
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ROŽMAN, Primož, JERAS, Matjaž, DOVČ, Tadeja, URBAJS, Matjaž, BOHINJEC, Mateja, and PRETNAR, Jože
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- 1998
28. The role of in vitro alloreactive T-cell functional tests in the selection of HLA matched and mismatched haematopoietic stem cell donors
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Jeras, Matjaž
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- 2002
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29. Applicability of human osteoarthritic chondrocytes for in vitro efficacy testing of anti-TNFα drugs
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Žigon-Branc, Sara, Jeras, Matjaž, Blejec, Andrej, and Barlič, Ariana
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- 2017
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30. Immunogenicity of dendritic cells pulsed with cartilage derived soluble alloantigens
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Maličev, Elvira, Knežević, Miomir, and Jeras, Matjaž
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- 2001
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31. Protein synthesis of human articular chondrocytes cultured in vitro for autologous transplantation
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Velikonja, Nevenka Kregar, Wozniak, Gordana, Maličev, Elvira, Knežević, Miomir, and Jeras, Matjaž
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- 2001
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32. The contribution of HLA-Class II antigens in humoral non-response and delayed response to HBsAg vaccination
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Vidan-Jeras, Blanka, Brinovec, Vlado, Jurca, Bojan, Stezinar, Snežna Levičnik, Jeras, Matjaž, and Bohinjec, Mateja
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- 2000
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33. In vitro functional tests for evaluation of stimulating capacity of cultured human dendritic cells
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Hajdinjak, Tine, Jeras, Matjaž, Knežević, Miomir, and Štabuc, Borut
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- 2000
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34. Testing the potency of anti‐TNF‐α and anti‐IL‐1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor‐matched chondrogenically differentiated mesenchymal stem cells.
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Žigon‐Branc, Sara, Barlič, Ariana, Knežević, Miomir, Jeras, Matjaž, and Vunjak‐Novakovic, Gordana
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CARTILAGE cells ,SPHEROIDIZING (Heat treatment) ,RHEUMATOID arthritis ,MACROPHAGES ,MESENCHYMAL stem cells - Abstract
Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor‐alpha (TNF‐α) and interleukin 1β (IL‐1β). New in vitro testing systems are needed to evaluate efficacies of new anti‐inflammatory biological drugs, ideally in a patient‐specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti‐inflammatory drugs, spheroids were exposed to TNF‐α, IL‐1β, or to supernatant containing secretome from activated macrophages (MCM). The anti‐inflammatory efficacies of anti‐TNF‐α biologicals adalimumab, infliximab, and etanercept, and the anti‐IL‐1β agent anakinra were assessed in short‐term microspheroid and long‐term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF‐α or IL‐1β. The differences in potency of anti‐TNF‐α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short‐term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti‐TNF‐α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045–1058, 2018 [ABSTRACT FROM AUTHOR]
- Published
- 2018
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35. Platelet gel stimulates proliferation of human dermal fibroblasts in vitro
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Domanovič, Dragoslav, Jeras, Matjaž, Krašna, Metka, Švajger, Urban, and Tomšič, Ana
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integumentary system - Abstract
Background: Platelet gel is a source of many soluble proteins that can modulate tissue regeneration. Dermal fibroblasts play an important role in tissue remodeling and wound healing. Objective: The study was performed to investigate the influence of platelet gel and its active ingredients on the proliferation of human dermal fibroblasts in vitro. Methods: A fibroblast culture was established from a normal human skin biopsy. Platelet gel was prepared from platelet-rich plasma (PRP) following the addition of calcified thrombin solution. cell proliferation was measured by MTT assay. Results: We showed that platelet gel, PRP, and thrombin stimulate the proliferation of dermal fibroblasts, and that stimulation by PRP is dose dependent. Conclusion:Platelet gel can be used to treat chronic skin defects.
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- 2007
36. Different localisation of cystatin C in immature and mature dendritic cells
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Bergant Marušič, Martina, Griffiths, Gareth, Jeras, Matjaž, and Zavašnik-Bergant, Tina
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- 2006
37. Analysis of Glioblastoma Patients' Plasma Revealed the Presence of MicroRNAs with a Prognostic Impact on Survival and Those of Viral Origin.
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Herman, Ana, Gruden, Kristina, Blejec, Andrej, Podpečan, Vid, Motaln, Helena, Rožman, Primož, Hren, Matjaž, Zupančič, Klemen, Veber, Matija, Verbovšek, Urška, Lah Turnšek, Tamara, Porčnik, Andrej, Koršič, Marjan, Knežević, Miomir, and Jeras, Matjaž
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GLIOBLASTOMA multiforme ,MICRORNA genetics ,PLETHORA (Pathology) ,TUMOR markers ,GLIOBLASTOMA multiforme treatment ,GENE expression ,PROGNOSIS - Abstract
Background: Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions. Experimental Procedures: MicroRNA profiling of 734 human and human-associated viral miRNAs was performed on blood plasma samples from 16 healthy individuals and 16 patients with GBM, using the nCounter miRNA Expression Assay Kits. Results: We identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients’ survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM. Conclusion: The GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients. [ABSTRACT FROM AUTHOR]
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- 2015
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38. Osteoimmunology and the influence of pro-inflammatory cytokines on osteoclasts.
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Zupan, Janja, Jeras, Matjaž, and Marc, Janja
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IMMUNE system , *BONE physiology , *BONE cells , *PROGENITOR cells , *BONE marrow physiology , *PHYSIOLOGICAL effects of cytokines , *NF-kappa B , *OSTEOCLASTS , *PHYSIOLOGY - Abstract
Bone and immune system are functionally interconnected. Immune and bone cells derive from same progenitors in the bone marrow, they share a common microenvironment and are being influenced by similar mediators. The evidence on increased bone resorption associated with inappropriate activation of T cells such as during inflammation, is well established. However, the molecular mechanisms beyond this clinical observation have begun to be intensively studied with the advancement of osteoimmunology. Now days, we have firm evidence on the influence of numerous proinflammatory cytokines on bone cells, with the majority of data focused on osteoclasts, the bone resorbing cells. It has been shown that some proinflammatory cytokines could possess osteoclastogenic and/or anti-osteoclastogenic properties and can target osteoclasts directly or via receptor activator of nuclear factor kB (RANKJ/RANK ligand(RANKL)/osteoprotegerin (OPG) system. Several studies have reported opposing data regarding (anti)osteoclastogenic properties of these cytokines. Therefore, the first part of this review is summarizing current evidence on the influence of pro-inflammatory cytokines on osteoclasts and thus on bone resorption. In the second part, the evidence on the role of pro-inflammatory cytokines in osteoporosis and osteoarthritis is reviewed to show that unravelling the mechanisms beyond such complex bone diseases, is almost impossible without considering skeletal and immune systems as an indivisible integrated system. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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39. Anti-inflammatory Effects of Resveratrol and Its Potential Use in Therapy of Immune-mediated Diseases.
- Author
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Švajger, Urban and Jeras, Matjaž
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RESVERATROL , *PHYTOCHEMICALS , *DISEASES , *IMMUNOPATHOLOGY , *IMMUNITY - Abstract
A molecule with a relatively simple chemical structure, resveratrol has been found to interact with multiple molecular targets, many of them associated with inflammation and immunity. Indeed, it has been shown to act directly on central players of both innate and adaptive immunity, such as macrophages, lymphocytes, and dendritic cells. In addition, there is very little evidence suggesting significant deleterious side effects of resveratrol, further highlighting its potential future use as a therapeutic agent. This review provides an up-to-date discussion on recent advances regarding anti-inflammatory effects of resveratrol, mechanisms of action, and its potential for therapeutic use. [ABSTRACT FROM AUTHOR]
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- 2012
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40. A "Crossomics" Study Analysing Variability of Different Components in Peripheral Blood of Healthy Caucasoid Individuals.
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Gruden, Kristina, Hren, Matjaž, Herman, Ana, Blejec, Andrej, Albrecht, Tanja, Selbig, Joachim, Bauer, Chris, Schuchardt, Johannes, Or-Guil, Michal, Zupančič, Klemen, Švajger, Urban, Štabuc, Borut, Ihan, Alojz, Kopitar, Andreja Nataša, Ravnikar, Maja, Kneževic, Miomir, Rožman, Primož, and Jeras, Matjaž
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BLOOD ,IMMUNOTHERAPY ,CAUCASIAN race ,CANCER treatment ,T cells ,BODY mass index - Abstract
Background: Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable. Methodology/Principal Findings: To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex ''crossomics'' analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4
+ T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset. Conclusions/Significance: Our study shows that the intra-individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective. [ABSTRACT FROM AUTHOR]- Published
- 2012
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41. Optimal Dendritic Cell Differentiation in RPMI Media Requires the Absence of HEPES Buffer.
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Švajger, Urban and Jeras, Matjaž
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DENDRITIC cells , *CELL differentiation , *MONOCYTES , *GENETIC regulation , *GENE expression , *MORPHOLOGY - Abstract
Monocyte-derived dendritic cells (DCs) are considered an indispensible and one of primary tools for in vitro DC-based studies. For majority of in vitro DC-based studies the medium of choice is supplemented RPMI, with certain variable ingredients such as HEPES buffer or Phenol Red (PHR). In effort to identify potential obstruction of DC differentiation process due to presence of mentioned additives, we differentiated DCs using RPMI either with or without HEPES or PHR. Although PHR caused a certain down-regulation of immature DCs (iDCs) differentiation markers and lower expression of co-stimulatory molecules on mature DCs, these changes were not significant. In contrast, use of RPMI also containing HEPES resulted in significantly lower CD1a and DC-SIGN expression on iDCs and extensively lowered co-stimulatory molecule expression after DC activation (HEPES-DCs). Furthermore, DCs differentiated in HEPES-free RPMI possessed more genuine immature//mature DC characteristics in context of Th1 polarization. Additionally, during classical differentiation procedure, fewer DCs remained adherent and possessed better overall morphology in HEPES-free medium. In summary our study clarifies a seemingly minor, but a very important issue, that will most likely facilitate lab work for many scientists dealing with monocyte-derived DCs. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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42. Novel Findings in Drug-Induced Dendritic Cell Tolerogenicity.
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Švajger, Urban, Obermajer, Nataša, and Jeras, Matjaž
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DENDRITIC cells ,ANTIGENS ,CELLS ,IMMUNE response ,IMMUNITY ,BIOCHEMICAL mechanism of action - Abstract
Dendritic cells (DCs) constitute a unique set of antigen-presenting cells (APCs), equipped with the potential to initiate strong immune responses as well as to critically regulate immunity. Tolerogenic or 'alternatively activated' DCs show remarkable properties in regulating immune responses both in vitro and in vivo. Furthermore, tolerogenic DCs are now beginning to be tested in clinical studies. Use of pharmacological agents to induce maturation-resistant tolerogenic DCs is a popular approach. In this review, we will discuss already recognized, as well as recently discovered, potential pharmacological agents, their mechanism of action, and the way in which they induce tolerance in DCs. [ABSTRACT FROM AUTHOR]
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- 2010
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43. Infuence of MHC on odour perception of 43 chemicals and body odour.
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Janeš, Damjan, Klun, Irena, Vidan-Jeras, Blanka, Jeras, Matjaž, and Kreft, Samo
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- 2010
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44. Induction/Engineering, Detection, Selection, and Expansion of Clinical-Grade Human Antigen-Specific CD8+ Cytotoxic T Cell Clones for Adoptive Immunotherapy.
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Jeras, Matjaž, Bricl, Irena, Robert Zorec, and Švajger, Urban
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CELL-mediated cytotoxicity , *T cells , *IMMUNOTHERAPY , *CLONE cells , *CELLULAR immunity - Abstract
Adoptive transfer of effector antigen-specific immune cells is becoming a promising treatment option in allogeneic transplantation, infectious diseases, cancer, and autoimmune disorders. Within this context, the important role of CD8+ cytotoxic T cells (CTLs) is objective of intensive studies directed to their in vivo and ex vivo induction, detection, selection, expansion, and therapeutic effectiveness. Additional questions that are being addressed by the scientific community are related to the establishment and maintenance of their longevity and memory state as well as to defining critical conditions underlying their transitions between discrete, but functionally different subtypes. In this article we review and comment latest approaches and techniques used for preparing large amounts of antigen-specific CTLs, suitable for clinical use. [ABSTRACT FROM AUTHOR]
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45. C-type lectin DC-SIGN: An adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity
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Švajger, Urban, Anderluh, Marko, Jeras, Matjaž, and Obermajer, Nataša
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LECTINS , *DENDRITIC cells , *IMMUNOREGULATION , *CELLULAR signal transduction , *CELL adhesion molecules , *ANTIGEN presenting cells , *MOLECULAR immunology , *ACETYLTRANSFERASES - Abstract
Abstract: The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a type II C-type lectin whose expression is restricted to the most potent antigen-presenting cells (APCs), the dendritic cells (DCs). In recent years, DC-SIGN has gained an exponential increase in attention because of its involvement in multiple aspects of immune function. Besides being an adhesion molecule, particularly in binding ICAM-2 and ICAM-3, it is also crucial in recognizing several endogenous and exogenous antigens. Additionally, the intracellular domain of DC-SIGN includes molecular motifs, which enable the activation of signal transduction pathways involving Raf-1 and subsequent modulation of DC-maturation status, through direct modification of nuclear factor Nf-κB in DCs. Upon DC-SIGN engagement by mannose- or fucose-containing oligosaccharides, the latter leads to a tailored Toll-like receptor signalling, resulting in an altered DC-cytokine profile and skewing of Th1/Th2 responses. In this article, we will discuss recent advances on a broad perspective concerning DC-SIGN structure, signalling and immune function. [Copyright &y& Elsevier]
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- 2010
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46. Preliminary evaluation of mRNA as as versatile vaccination platform against infectious diseases
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Jug, Špela and Jeras, Matjaž
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udc:614.47:615.37(043.3) ,mRNA adjuvanti limfocitni horiomeningitis intranodalna imunizacija imunska terapija ,immunotherapy vaccination mRNA adjuvants viruses HIV-1 LCMV influenza A virus - Abstract
Infectious diseases kill millions of people every year. Despite quickly evolving technology, vaccination remains the most efficient way to prevent and treat them in most regions of the world. Vaccines are based on the immunological memory of adaptive immune system. For achieving the most effective response of this part of immune system, a prior activation of innate immunity is required. In this process, dendritic cells (DC) represent the main connecting link between the two systems. DCs are professional antigen presenting cells (APCs), capable of antigen acquiring, processing and presenting it to T cells. Because of their important position in the activation of adaptive immune system, DCs represent an important target model for vaccine production. So far, most clinical studies have used these APCs for specific activation of cytotoxic T cells (CTLs), which are the only cells being able to completely eliminate viral infection. Recently, there a growing interest in using mRNA as a vaccine platform to activate DCs has evolved, mainly due to its favorable safety profile and ability to efficiently activate cell-mediated immune responses. Actually, mRNA-vaccines offer a compelling alternative in preventing and treating various infections, as they can be rather quickly produced, are transiently expressed and suitable for encoding different antigens. For these reasons, we set up to evaluate feasibility and efficiency of intranodal immunization of mice with mRNA-based vaccines in evoking antigen-specific T and B cell immune responses. For our experiments we used three mouse-models, HIV-1, LCMV and influenza A virus. Depending on the model, mRNAs were encoded with different viral antigens (HIVACAT, NP, gp and HA) and adjuvants (TriMix: CD40L, CD70 and caTLR4 DiMix: CD40L and caTLR4 and flagellin). We demonstrated that intranodal immunization of mice with mRNA-vaccines can generate strong antigen-specific T-cell and B-cell immune responses. With a concomitant application of the TriMix mRNA we achieved stronger CTL responses following the first and the second, but not after the third immunization. In contrast, flagellin enhanced humoral immune responses to influenza hemagglutinin (HA) but turned to be immunosuppressive in regard of CTL activation. Our preliminary experimental results are only a small contribution to the complex field of mRNA-vaccines development, but they suggest that by continuing research in this direction the efficiency of fighting various types of life-threatening diseases could be improved in future. Kljub hitremu tehnološkemu napredku na številnih podočjih ostaja cepljenje najučinkovitejši pristop za preprečevanje infekcijskih bolezni, ki letno terjajo milijone življenj po svetu. Cepiva temeljijo na imunskem spominu celic pridobljene imunosti. Za popoln oz. obsežen odziv tega dela imunoskega sistema pa je potrebna ustrezna predhodna aktivacija celic prirojene imunosti. Pri tem predstavljajo dendritične celice (DC) poglavnitno povezavo med naravno in pridobljeno imunostjo. Gre za profesionalne antigene predstavitvene celice (APC), sposobne privzemati antigenske proteine, jih razgraditi v peptide in nato, vezane na molekule poglavitnega kompleksa tkivne skladnosti (MHC), predstaviti T-celičnim receptorjem na limfocitih T. Potem ko se antigenski peptide vežejo na molekule MHC razreda I, jih lahko prepoznajo T-celični receptorji (TCR), izraženi na določenih klonih citotoksičnih CD8+ limfocitov T (CTL), ki imajo osrednjo vlogo pri odstranjevanju z virusi okuženih in rakavo spremenjenih celic. Antigenske peptide, ki se vežejo na molekule MHC razreda II, pa specifično prepoznavajo TCR, prisotni na ustreznih klonih CD4+ T celic pomagalk (celice Th). Celice Th so pomembne tako za dozorevanje in proliferacijo ter okrepitev delovanja CTL (preko DC), kot tudi za proizvodnjo visoko afinitetnih antigensko specifičnih protiteles v plazmatkah. Zaradi velikega vpliva, ki ga imajo DC na pridobljeni imunski odziv, predstavljajo pomemben model za izdelavo cepiv. Do sedaj so jih v večini kliničnih testiranj uporabili z namenom aktivacije imunskega sistema pri infekcijskih boleznih in različnih oblikah raka. Cilj je bil predvsem aktivacija specifičnih citotoksičnih odzivov CLT, saj lahko le ti popolnoma odstranijo z virusi okužene ali rakavo spremenjene celice. V zadnjem času se je zaradi njenega varnostnega profila in sposobnosti aktivacije celičnega imunskega odziva močno povečalo zanimanje za uporabo mRNA-cepiv. V primerjavi z drugimi dostavnimi sistemi antigenov se je namreč ta, zaradi svoje prehodne ekspresije v citosolu, možnosti vkodiranja različnih antigenskih proteinov ter hitre izdelave, pokazal kot zanimiva možnost v boju proti različnim povzročiteljem okužb. Namen naše magistrske naloge je bil preučiti izvedljivost in učinkovitost intranodalne imunizacije z mRNA, kodirane z različnimi antigeni in adjuvanti, s ciljem aktivacije antigensko specifičnih T- in B- celičnih imunskih odzivov pri različnih okužbah in vivo. Dokazali so namreč, da so DC v bezgavkah sposobne privzemati mRNA, izraziti kodirane antigenske proteine, jih v obliki peptidov predstaviti v kontekstu molekul MHC in uspešno aktivirati celični imunski odziv. Pri našem delu smo uporabili 3 mišje modele, in sicer HIV-1, LCMV in influenca A. V mRNA smo vkodirali različne antigene, glede na uporabljeni model, in sicer skupaj z različnimi adjuvanti TriMix (CD40L, CD70, caTLR4), DiMix (CD40L, caTLR4) in flagelin. Adjuvante smo uporabili zato, ker določajo učinkovitost cepiva oz. njegovo sposobnost aktivacije DC. Mišji model virusa človeške imunske pomanjkljivosti 1 (HIV-1) Razvoj protiretrovirusnega zdravljenja v zadnjih letih je zagotovil novo upanje za osebe, ki so okužene s HIV-1. Kljub učinkovitosti pa tovrstno zdravljenje ni kurativno, zato je vseživljenjsko, spremljajo pa ga pogosti neželeni učinki, bolniki pa so tudi bolj dovzetni za srčnožilne in nevrološke bolezni. Zaradi osrednje vloge imunskega sistema pri okužbi z virusom HIV-1, bi lahko bila imunoterapija pomembna nova metodo za premagovanje te bolezni. Odločili smo se, da bomo v mRNA kodirali ohranjena strukturna področja virusa HIV-1 (struktura HIVACAT), za katere je znano, da so tarče T-celičnega imunskega odziva v obolelih osebah z nizko plazemsko koncentracijo virusa. V bezgavkah in vranici poskusnih živalih smo, po prvi, drugi in tretji imunizaciji s cepivoma HIVACAT mRNA in HIVACAT mRNA + TriMix, ugotavljali obseg specifičnega citotoksičnega odziva CD8+ limfocitov T. Ugotovili smo, da je vsaka imunizacija povečala omenjeni imunski odziv. Prisotnost adjuvanta TriMix je pripomogla k močnejšemu odzivu CTL po prvi in drugi imunizaciji, v primerjavi s skupino živali, ki je prejela cepivo brez njega. Po tretji aplikaciji pa so razlike med primerjanima skupinama postale manjše. Ob tem smo preverili tudi morebiten vpliv strukture TriMix na obseg in jakost T-celičnih odzivov na 17 strukturnih področij HIV-1, kodiranih v strukturi HIVACAT, pri čemer nismo opazili večjih razlik, v primerjavi s skupino živali, ki je prejela zgolj cepivo HIVACAT mRNA. ii) Mišji model virusa limfocitnega horiomeningitisa (LCMV). Mišji model LCMV se zaradi podobnega vpliva na imunski sistem, kot ga ima HIV-1, pogosto uporablja za odkrivanje imunskih lastnosti slednjega. Dva poglavitna strukturna proteina v LCMV, ki sta odgovorna za citotoksični T-celični imunski odziv nanj, sta nukleoprotein in glikoprotein. Zato smo v mRNA vkodirali oba, in sicer v kombinaciji z različnimi adjuvantnimi imunostimulatornimi molekulami (TriMix, DiMix in flagelin). Analizirali smo jakost imunskih odzivov CTL na oba antigenska proteina, in sicer glede na dodani adjuvant. V vseh primerjanih skupinah poskusnih živali smo določili močne odzive, s čimer smo potrdili visoko imunogenost obeh antigenskih proteinov (nukleoproteina in glikoproteina). Ugotovili smo, da je bil pri tem TriMix najučinkovitejši adjuvant, flagelin pa je deloval imunsupresivno na aktivacijo CTL. Mišji model virusa influence A Na koncu smo preverili, kakšen vpliv imajo mRNA-cepiva na humoralni imunski odziv. Želeli smo ugotoviti, ali intranodalna imunizacija z mRNA lahko izzove protitelesni imunski odziv proti hemaglutininu virusa influence A, in ali ga lahko z dodatkom adjuvantov še povečamo. V plazmi imuniziranih poskusnih živali smo skozi daljše časovno obdobje določali protitelesa IgG1 in IgG2a. Medsebojno smo primerjali tudi različne skupine živali, ki so prejele cepivo z različnimi adjuvanti (TriMix, DiMix in flagelin). Najučinkovitejši adjuvant je bil flagelin, TriMix pa je zaviral na humoralni imuski odziv. Za podrobnejšo analizo smo uporabili različne kombinacije imunostimulatornih adjuvantnih molekul (CD40L, CD70, caTLR4, flagelin), da bi lahko ocenili vpliv posamezne od njih. Tiste skupine živali, pri katerih je bil v cepivu prisoten flagelin, so izkazale obsežnejše B-celične oz. humoralne imunske odzive glede na tiste, pri katerih tega adjuvanta nismo uporabili. Nasprotno pa smo pri živalih, pri katerih so bile v uporabljenem cepivu kodirane adjuvantne molekule CD70, opazili njihov imunosupresiven učinek na B-celični imunski odziv. Dokazali smo torej, da intranodalna imunizacija z mRNA-cepivi izzove T- in B-celične imunske odzive. S sočasno uporabo adjuvanta TriMix smo dosegli povečanje citotoksičnega odziva CD8+ limfocitov T po prvi in drugi, ne pa tudi po tretji imunizaciji. Flagelin je povečal humoralni odziv, na T celični odziv pa je deloval imunosupresivno. Naši preliminarni eksperimentalni rezultati predstavljajo le majhen prispevek v okviru kompleksnega razvoja mRNA-cepiv, vandar pa nakazujejo, da bi lahko z nadaljnjih delom v tej smeri pripomogli k učinkovitejšem boju proti različnim vrstam življenja ogrožajočih okužb.
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- 2020
47. Properties of human embryonic stem cells with 18q deletion in cell culture and during their in vitro differentation
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Krivec, Nuša and Jeras, Matjaž
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human embryonic stem cells chromosome instability 18q deletion mosaicism ,udc:576.3+611.013(043.3) ,človeške embrionalne matične celice kromosomska nestabilnost delecija 18q mozaicizem mozaične kulture - Abstract
Človeške embrionalne matične celice (EMC) so pluripotentne celice, ki imajo sposobnost samoobnavljanja in diferenciacije v katerikoli celico odraslega človeškega telesa. Embrionalne matične celice lahko uporabljamo na področju farmacevtskih raziskav, celičnih terapij in presaditev tkiv in organov. Najpogosteje jih pridobivamo z izolacijo iz notranjega celičnega skupka blastociste embrijev, pridobljenih s postopkom oploditve in vitro. Leta 2006 sta Takahashi and Yamanaka uspešno reprogramirala somatske celice v pluripotentne matične celice z uporabo genov za transkripcijske dejavnike Oct4, Sox2, Klf4 and c-Myc, ki se močno izražajo v pluripotentnih EMC. Za gojenje EMC v in vitro kulturi so sprva uporabljali hranilne plasti mišjih embrionalnih fibroblastov (MEF) in gojišče z dodanimi rasnimi dejavniki in govejim serumom. Ta standardni protokol vsebuje veliko živalskih produktov, ki lahko vsebujejo imunogene in bi pri klinični uporabi predstavljali nevarnost za reakcijo z imunskim sistemom prejemnika. Razvoj gre v smeri uporabe pogojev brez hranilnih plasti in gojišč brez seruma. Večinoma se uporabljajo ekstracelularni matriksi, kot so Matrigel, kolagen IV, laminin, fibronektin in gojišča s humanim serumom ali njegovimi nadomestki. Embrionalne in inducirane pluripotentne matične celice lahko med in vitro kulturo pridobijo kromosomske nepravilnosti, ki jim omogočajo selektivno prednost in s tem razrast v celični kulturi. Najpogostejše kariotipske spremembe vključujejo pridobitve na kromosomih 1, 12, 17, 20 in X. Te mutacije so lahko posledica anafaznega zaostajanja, kromosomskega nerazdvajanja in poškodb DNA. Spits in sodelavci (2008) so prvi poročali o deleciji na dolgem kraku kromosoma 18. Delecija se je v celicah pojavila skupaj z duplikacijo na kromosomu 5q ali 7q. Kot posledica nepravilne poprave je nastal derivativni kromosom 18, ki je vseboval del kromosoma 18 in del kromosoma 5 ali 7. Na lokusu 18q je prisotnih veliko tumor-supresorskih genov, kot so SMAD4, SMAD2 in DCC. V tej magistrski nalogi smo spremljali človeške EMC z delecijo 18q v kulturi in med diferenciacijo. Najprej smo želeli poiskati označevalec, ki bi omogočil razlikovanje med celicami mutanta in divjega tipa. Naš cilj je bil raziskati, ali nediferencirane celice z delecijo 18q, ki so gojene na Lamininu-521, pridobijo selektivno prednost, kot se to zgodi v kokulturi z MEF. Zanimale so nas tudi lastnosti mozaičnih kultur med spontano in usmerjeno diferenciacijo v hepatoblast. Mozaične kulture smo ustvarili z mešanjem različnih deležev celic divjega tipa (DT) in mutanta (MT) (10 % MT z 90 % DT in 50 % MT z 50 % DT). Za razlikovanje med celicami DT in MT smo uporabili metodo verižne reakcije s polimerazo v realnem času, s testom za določanje variabilnega števila kopij genov ATP9B in NFATC1 in testom za ugotavljanje stopnje izražanja gena LINC00116. Na nivoju proteinov smo uporabili protitelesa proti citoplazemskemu proteinu TSPYL5, ki smo jih zaznavali z imunofluorescenčno mikroskopijo, avtomatskim celičnim števcem in pretočnim citometrom. Preverili smo občutljivost testov in njihovo zmožnost zaznavanja celic mutanta v mozaičnih kulturah. Pripravili smo mešanice DNA in RNA z različnim deležem DT in MT. Rezultati so pokazali, da lahko spremembo v številu kopij in izražanju gena zanesljivo zaznamo v primeru, da je razlika v vsebnosti mutanta vsaj 20 %. Mozaične kulture nediferenciranih celic smo vzdrževali v in vitro pogojih 13 pasaž, med katerimi smo izmenično določali izražanje in število kopij genov. Količina mutanta je med posameznimi pasažami nihala, vendar pa v nobenem primeru ni prišlo do razrasta mutanta v kulturi. Najbolj se je prevzetju približala kultura s 50 % začetno vsebnostjo mutanta celične linije VUB04_CF, saj je količina mutanta narasla v prvih treh pasažah in ostala precej konstantna do konca poskusa. Dokazali smo tudi, da je pritrjevanje celic na laminin enako učinkovito tako pri divjem tipu kot pri mutantu in da se razmerje med njima ne spreminja na podlagi tega dejavnika. Po odvzemu dejavnika, ki vzdržuje pluripotentnost celic, začnejo EMC spontano diferencirati, kar vpliva na signalne poti in celični cikel. Drugačen profil izražanja genov celic z delecijo 18q lahko privede do prednosti pred divjim tipom v procesu specializacije celic. Spremembe v našem eksperimentu so se pokazale tako na fenotipu kot na znižanju števila kopij in znižanju izražanja gena ter s tem dokazale selektivno prednost mutanta. Enako smo pričakovali tudi pri diferenciaciji celic v hepatoblast, saj so izpostavljene stresnim dejavnikom med diferenciacijo v specifičen tip celic. Izkazalo se je, da celice z mutacijo na kromosomu 18 ne pridobijo prednosti v kulturi med usmerjeno diferenciacijo v hepatoblast. Na nivoju proteinov smo želeli razliko med divjim tipom in mutantom pokazati z uporabo protitelesa proti proteinu TSPYL5. Z barvanjem jeder z barvilom Hoechst in analizo s pretočnim citometrom smo uspeli pokazati spremembe v celičnem ciklu med diferenciacijo, medtem ko je bilo razlikovanje med divjim tipom in mutantom neuspešno, saj se je protitelo vezalo tako v citoplazmi divjega tipa kot mutanta pred in po diferenciaciji. Po podatkih iz literature je meja zaznave mozaicizma z metodo qPCR med 5 % in 10 %. Z našimi testi za število kopij in izražanje gena smo razliko med MT in DT zaznali pri spremembi količine MT za 20 %, kar je bilo za naše eksperimente sprejemljivo. Mešanice enakih deležev MT in DT so ustvarile enake pogoje za preučevanje selektivne prednosti, medtem ko so mešanice z 10 % MT služile kot realistični primeri naravnega mozaicizma, pri katerih bi lahko zaznali prevzem kulture. Po predhodnih podatkih so celice z delecijo 18q prevzele kulturo v prvih nekaj pasažah, v primeru gojenja celic na hranilni plasti MEF, medtem ko v kulturi na Lamininu-521 razrasta mutanta nismo opazili. Razlog je lahko v uporabi različnih tehnik disociacije pri pasažah. Za sproščanje celic z laminina smo uporabili encime in tako pridobili suspenzijo celotne populacije celic v gojilni posodi, vključno s poškodovanimi in senscentnimi celicami. Kolonije v kulturi na hranilni plasti pa je izbral operator, jih mehansko razrezal na majhne kose in jih prenesel v novo gojilno posodo. Pri tej tehniki obstaja večja možnost pristranskosti pri izbiri kolonij, ki lahko vpliva na količino mutanta v novi pasaži. Pri spontani diferenciaciji preživi več celic z delecijo 18q kot celic divjega tipa. Na kromosomu 18q se nahajajo številni tumor-supresorski geni, kar lahko pojasni selektivno prednost celic z delecijo. Proces diferenciacije in manj tumor-supresorskih genov v mutantu bi lahko sprožili maligno transformacijo. Med usmerjeno diferenciacijo so celice izpostavljene večjemu selekcijskemu pritisku, saj dodani faktorji, ki sprožijo diferenciacijo, vplivajo na spremembe v signalnih poteh. Čeprav smo pričakovali, da bodo rezultati podobni kot pri spontani diferenciaciji, je razmerje med divjim tipom in mutantom ostalo enako, kar je možno pojasniti s tem, da spremembe v bioloških procesih med usmerjeno diferenciacijo v hepatoblast ne vplivajo na celice z delecijo 18q in njihov molekulski profil ni pomemben za razvoj endoderma. Glede na rezultate analize izražanja gena TSPYL5 smo pričakovali, da bo protein, ki ga gen kodira, ustrezen označevalec celic z delecijo 18q, vendar se je izkazal kot neuspešen. Številni dejavniki, kot so posttranskripcija, translacija in hitrost razgrajevanja proteina, vplivajo na povezavo med RNA in proteini ter lahko povzročajo razlike v izražanju. S to raziskavo smo dobili vpogled v lastnosti EMC z delecijo 18q, vendar so za potrditev rezultatov in za boljšo statistično značilnost potrebni dodatni poskusi z več biološkimi ponovitvami. Human embryonic stem cells (hESCs) are pluripotent cells that possess properties of self-renewal and the capacity to differentiate into any cell type of the adult human body. Embryonic stem cells are valuable for their use in pharmaceutical research, cell therapies and transplantation procedures. However, when hESCs are kept in in vitro culture, they tend to acquire chromosomal abnormalities, which, in case they provide the hESCs with a selective advantage, can cause aberrant cells that can take over the culture. One of the reported abnormalities that confer a selective advantage to cells when co-cultured with mouse embryonic fibroblasts (MEFs), is a derivative chromosome 18. In this thesis we focused on progression of the 18q deletion during undifferentiated hESCs culture and upon their differentiation, when cultured on a more clinically-relevant feeder-free cell culture system based on Laminin-152. Mosaic cultures were created by mixing different ratios of wild-type (WT) and mutant (MT) cells (10% of MT and 90% of WT, 50% of MT and 50% of WT). Real time polymerase chain reactions, with copy number assays for genes ATP9B and NFATC1 and gene expression assays for LINC00116 were used to discriminate between wild-type and mutant cells in mixes. At the protein level, antibodies against cytoplasmic protein TSPYL5 were used. They were detected with immunofluorescent microscopy, an automated cell counter and flow cytometry. Genes ATP9B, NFATC1 and LINC00116 are suitable genomic markers for cells carrying the 18q deletion, but protein marker TSPYL5 failed to distinguish between the WT and MT cells. In the culture of undifferentiated hESCs, we observed fluctuations in ratios of MT vs WT cells. However, partial take-over was only observed in the culture containing 50% MT of one cell line, while in all other combinations there was no significant increase observed between passages. In contrast to spontaneous differentiation, where cells with 18q deletion seem to display a selective advantage upon directed hepatoblast differentiation, the ratios of WT and MT cells remained similar. Further research should be carried out to confirm and validate our results.
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- 2020
48. Mechanism of Endosomal TLR Inhibition by Antimalarial Drugs and Imidazoquinolines.
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Kužnik, Alenka, Bencina, Mojca, Švajger, Urban, Jeras, Matjaž, Rozman, Blaž, and Jerala, Roman
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SYSTEMIC lupus erythematosus treatment , *ANTIMALARIALS , *IMMUNE response , *AUTOIMMUNITY , *CHLOROQUINE - Abstract
Endosomal TLRs play an important role in innate immune response as well as in autoimmune processes. In the therapy of systemic lupus erythematosus, antimalarial drugs chloroquine, hydroxychloroquine, and quinacrine have been used for a long time. Their suppression of endosomal TLR activation has been attributed to the inhibition of endosomal acidification, which is a prerequisite for the activation of these receptors. We discovered that chloroquine inhibits only activation of endosomal TLRs by nucleic acids, whereas it augments activation of TLR8 by a small synthetic compound, R848. We detected direct binding of antimalarials to nucleic acids by spectroscopic experiments and determined their cellular colocalization. Further analysis revealed that other nucleic acid-binding compounds, such as propidium iodide, also inhibited activation of endosomal TLRs and colocalized with nucleic acids to endosomes. We found that imidazoquinolines, which are TLR7/8 agonists, inhibit TLR9 and TLR3 even in the absence of TLR7 or TLR8, and their mechanism of inhibition is similar to the antimalarials. In contrast to bafilomycin, none of the tested antimalarials and imidazoquinolines inhibited endosomal proteolysis or increased the endosomal pH, confirming that inhibition of pH acidification is not the underlying cause of inhibition. We conclude that the direct binding of inhibitors to nucleic acids mask their TLR-binding epitope and may explain the efficiency of those compounds in the treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]
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- 2011
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49. Quantification of cell hybridoma yields with confocal microscopy and flow cytometry
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Gabrijel, Mateja, Repnik, Urška, Kreft, Marko, Grilc, Sonja, Jeras, Matjaž, and Zorec, Robert
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ANTIGENS , *FLOW cytometry , *CANCER cells , *VACCINES - Abstract
The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells. [Copyright &y& Elsevier]
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- 2004
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50. Assessment of potential protective biomarkers in serum and saliva samples of HIV-1 infected participants from Rwanda
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Mitrović, Sanja and Jeras, Matjaž
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sindrom pridobljene imunske pomanjkljivosti citokini zaščitna protitelesa ELISA metoda Luminex Multiplex ,udc:616.98:578.828(043.3) ,HIV cytokines protective antibodies ELISA Luminex Multiplex method - Abstract
Despite more than 30 years of scientific and medical efforts, HIV infection is still a major global public health concern. The HIV pandemic continues to expand, with an average of 2,5 million new infections per year. Sub-Saharan Africa, including Rwanda, remains the most affected region in the world, accounting for approximately 70% of all people living with HIV. Therefore, safe and effective HIV prevention methods for Africa are urgently needed. A number of studies have shown that the cytokine network plays a crucial role in the pathogenesis of HIV infection. This raises the question whether cytokines in HIV disease can be a target for new approaches to develop a safe vaccine. The aim of our work was the assessment of potential protective biomarkers in serum and saliva samples of 14 HIV-1 infected participants from Rwanda. For this purpose, we used two methods to analyze our samples, i.e. the in-house ELISA test for detection of IgG and IgA antibodies against HIV-Gag p24 and HIV-Env gp140 proteins, and the Luminex Multiplex technology for measuring 15 soluble proteins involved in the Th17 immune response pathway (TNF-α, IL-6, IL-1β, IL-10, IFN-γ, IL-4, IL-17A, IL-17F, IL-17E, sCD40L, IL- 21, IL-22, IL-23, IL-31 and IL-33). Our results showed statistically significant correlations between serum concentrations of IL-6 and IgA anti-EnvA, as well as IL-33 and IgG anti- EnvA. Additionally, we found statistically significant correlations in saliva samples for IL- 1β with IgG anti-EnvA, IL-4 with IgG anti-EnvA and IgG anti-p24, IL-17A with IgA antip24, IL-17E with IgG anti-p24, and IL-23 with IgG anti-EnvA. Taken together, our results suggested that there are correlations between specific HIV-1 antibody and cytokine concentrations, indicating some potential protective biomarkers in HIV infection (IL-6, IL- 33, IL-1β, IL-4, IL-17A, IL-17E and IL-23). However, implicating cytokines in the development of an HIV cure is not a simple task and several limitations of our results should be noted. Therefore, further studies on the cytokine protective role in HIV disease are necessary to clarify their role in the real clinical conditions. Okužba z virusom HIV, kljub več kot 30 letom boja proti njej, še vedno ostaja eden večjih javnozdravstvenih problemov na svetu. Epidemija okužb z virusom HIV narašča v obsegu približno 2,5 milijona novih primerov na leto. Podsaharska Afrika, vključno z Ruando, ostaja najbolj prizadeta regija na svetu, kjer število ljudi, ki so okuženi z virusom HIV, predstavlja približno 70% vseh HIV-pozitivnih posameznikov na svetu. Zato zanj nujno potrebujemo varno in učinkovito cepivo. Pri okužbi z virusom HIV imajo pomembno vlogo citokini, topne proteinske signalne molekule, ki so del imunskega sistema. Citokini bi lahko bili uporabni pri iskanju novih pristopov za razvoj učinkovitega cepiva proti HIV. Seveda pa moramo najprej razumeti njihovo delovanje pri okužbi z virusom. To področje so že intenzivno raziskali, pri tem pa dobili nasprotujoče si rezultate. V okviru magistrske naloge smo določali prisotnost potencialnih zaščitnih označevalcev v serumu in slini 14 posameznikov iz Ruande, okuženih z virusom HIV. Pri tem smo primerjali koncentracije izbranih citokinov s titri specifičnih protiteles proti HIV. Uporabili smo dve metodi, test ELISA za detekcijo protiteles IgG in IgA proti proteinom HIV-Gag p24 in HIV-Env gp140 ter tehnologijo Luminex Multiplex za merjenje koncentracij 15 citokinov, ki sodelujejo v imunskih poteh celic Th17 (TNF-α, IL-6, IL-1β, IL-10, IFN-γ, IL-4, IL-17A, IL-17F, IL- 17E, sCD40L, IL-21, IL-22, IL-23, IL-31 in IL-33). Dokazali smo statistično značilne povezave med serumskimi koncentracijami IL-6 in protiteles IgA proti EnvA ter IL-33 in protiteles IgG proti EnvA. Poleg tega smo z meritvami citokinov v vzorcih sline potrdili tudi statistično značilne povezave med IL-1β ter protitelesi IgG proti EnvA, IL-4 in protitelesi IgG proti EnvA ter protitelesi IgG proti p24, IL-17A in protitelesi IgA proti p24, IL-17E in protitelesi IgG proti p24 ter IL-23 in protitelesi IgG proti EnvA. Zaključimo lahko, da pri preiskovanih osebah, okuženih s HIV, obstajajo povezave med koncentracijami citokinov (IL-6, IL-33, IL-1β, IL-4, IL-17A, IL-17E in IL-23) in titri protiteles razredov IgG in IgA proti HIV-Gag p24 in HIV-Env gp140. Sistemska uporaba citokinov v klinični praksi je zelo zahtevna. Statistično značilne povezave, ki smo jih odkrili, zato predvsem nakazujejo smer, v katero bi lahko usmerili nadaljnje raziskave, ki so nujno potrebne za boljše razumevanje morebitne zaščitne vloge posameznih citokinov pri okužbi z virusom HIV.
- Published
- 2018
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