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Karaciğer rejenerasyonu: Mezenkimal kök hücre ve toll benzeri reseptörlerin potansiyel rolleri
Liver has unique capacity to regenerate in response to loss of hepatocytes depending on viral infections, toxic reactions and cancer formation. Although liver regeneration have been extensively studied, factors participate in the process are still under extensive investigation. Differentiation and immunosuppressive potential, as well as homing capacity to the site of injury of mesenchymal stem cells (MSCs) can be harnessed in liver regeneration. Moreover, toll like receptors (TLRs) which are known to involve immuneregulatory mechanisms of MSCs may also be a player in this process. Therefore, in this study, our aim was to understand the possible roles of MSCs and TLRs during liver regeneration. First, TLR message distribution upon partial hepatectomy induction was assessed. Then, homing capacity of MSCs in liver regeneration was examined by injecting labeled MSC generated from normal or already PH initiated rats back into partially hepatectomized (PH) rats. After investigating MSC homing, mRNA expression profiles of several key TLRs in the course of liver regeneration with or without MSC administration was examined. Finally, the role of TLRs in immunoregulatory properties of MSCs was explored. Our data showed that MSCs from normal rats and at day 1 post PH are localized in liver of PH generated animals. We have also determined that injected MSCs increased TLR2, 3 and 9 expressions in livers in comparison to that of hepatectomized liver that did not receive any MSC injection. Therefore, our data suggests that during liver regeneration at post PH day 3, homing of MSCs to the site of injury is at maximum and TLRs do not play any roles in recruiting these cells to liver, but rather localized MSCs either cis or at trans manner promote TLR expression. Despite the fact that MSCs are known to be non-immunogenic, in the present study isolated MSCs from BM are found to be expressing a panel of TLR mRNAs and our findings strongly implicate that these TLRs are functional in terms of cytokine secretion upon triggered by their proper ligands. Finally, we have identified that mouse MSC possesses different levels of critical surface markers such as CD11b, CD45, CD90 and CD117 at different passages and led us to think that either sub-populations or contaminating cell fractions may exist within the studied MSC population and furthermore may contribute to stimulatory potential of MSCs.
NK-92 hücrelerinin tümör antijenlerine yönlendirilmiş farklı kimerik anitjen reseptör tasarımlarının sistemik karşılaştırılması
Cancer immunotherapies focus on the power of the immune system to attack tumor cells. Recently, Chimeric Antigen Receptors expressing T cells (CAR-T cells) have received clinical approval for antigen-specific adoptive immunotherapy against CD19 in B cell malignancies. CAR vector designs have dramatically developed since their initial discovery and now include first-generation CARs (CD3ζ-based CAR), second-generation CARs with additional costimulatory domains such as CD28 or CD137 and third generation CARs (CD3ζ with two costimulatory domains) and recently fourth generation CAR with a transgene for cytokine stimulation. Natural Killer (NK) cells have ability recognize the tumor cells by their native receptors and have grown to be promising candidates for adoptive immunotherapy of cancer. CAR expression in NK cells is also clinically tested and carries the potential to translate into clinical application but the majority of literature on CAR vector design relies on observations from T cells. This thesis aims to use NK-92 cells for evaluation of different designs in order to optimize a CAR vector that could be efficiently used to retarget NK cells against tumor antigens. CAR transgenes comprising identical antigen binding domains that target CD19, combined with different intracellular signaling domains (CD3ζ , CD28 and CD137) are transferred to NK-92 cells via the use of lentiviral vectors. Cytotoxic activity and antigen-specificity of CAR-NK-92 cells are evaluated against the CD19- classical NK cell target K562 cell line and the CD19+ cell line Daudi and Namalwa by analysis of degranulation and cytokine secretion. Our results provide valuable data for optimal CAR vector design in NK cells.
Çeşitli tümör hücrelerine karşı genetik olarak değiştirilmiş NK-92 hücreleri kullanılarak yeni bir in vitro tarama yönteminin geliştirilmesi
Natural killer (NK) cells of the innate immune system are recognized for their ability to potently kill tumor cells. NK cell-mediated lysis is maintained by an intricate balance between several activating and inhibitory receptors that either trigger or dampen effector functions upon ligand engagement. In this study, we aim to dissect this complex balance by developing a cell-based screening tool to identify receptor specific anti-tumor responses. As the character of the heterogeneous tumor cell populations differs among patients, such a tool may be instrumental in developing patient-tailored cancer immunotherapies. Genes encoding 20 NK cell surface receptors were cloned into lentiviral vectors for genetic modification of the NK-92 cell line. Genetically modified (GM) NK-92 cells were enriched and overexpression of receptors was confirmed by flow cytometry. We analyzed the effector functions of all GM NK-92 cells against human cancer cell lines as well as against primary human sarcoma explants. Overall, genetic modifications did not hamper cytotoxic capacity of GM NK-92 cells; rather induced enhanced tumor cell targeting by receptors such as DNAM-1 and NKG2D. We further confirmed that this response was indeed DNAM-1 or NKG2D-dependent by using blocking antibodies. We also evaluated the synergistic response of prominent receptors in triggering degranulation and cytotoxicity by co-expressing DNAM-1 and NKG2D. Our results show the feasibility of an in vitro genetic screening approach to identify response-triggering receptors in genetically modified NK cells expressing different activating receptors. This tool has the potential to rapidly identify patient-specific targets for adoptive immunotherapy of cancer.
Lomber omurga sonlu eleman model çalışması ve lomber dinamik stabilizasyon sistemi analizi
Finite element (FE) method is a reliable analysis tool in mechanical engineering as well as in other fields such as electronics and thermodynamics. The use of FE models in biomechanics increased in last couple of decades, particularly in spine biomechanics, largely due to better computational resources available. However, development of spine FE model is a cumbersome and non-trivial task, owing to the complex geometry of the spinal segments. FE models have been used to produce general behaviors of the musculoskeletal systems with almost all physiological and anatomical features. The main objective of the present thesis is to develop accurate FE models of the human lumbar and thoracic spine. Lumbar FE models are particularly important for the clinicians as well as academicians. It is because low back pain constitute as the most prevalent disease of spine. A novel method was suggested in this thesis to construct an integrated interface between the discs and the vertebrae. The exact geometry was obtained from CT scan data. Multi-block method was used to place nodes over the vertebrae and intervertebral disc surfaces, and hexahedral element was used to mesh the discs and vertebrae. Truss elements were used to simulate the ligaments. Facet joints were simulated by unidirectional gap elements. Material properties were assigned to spinal components using the values from the literature. Pure moments were applied to a flying node which was coupled with top surface of first vertebrae. Lower part of last vertebrae in each model was constrained in all directions. The predicted motion response of FE model was compared with the published in vitro studies in all motion planes and found to be in good agreement. The validated FE model was used to study the biomechanical parameters of the lumbar spine in intact and instrumented cases. Various designs of instruments were applied to both the thoracic and the lumbar models. The effect of the instruments on the kinematic, i.e. range of motion (ROM), and kinetic, i.e. intradiscal pressure (IDP), facet load (FL), and ligament stress (LS), parameters of the lumbar spine and the thoracic spine were investigated. The lumbar instruments included posterior dynamic stabilization (PDS), fusion, interspinous fusion, and pedicle screws with spring rods. Design modifications were suggested after comparing the biomechanical behavior of the instrumented models with intact models.