Browsing by Author "Kahraman, Tamer"
Now showing 1 - 10 of 10
Results Per Page
Sort Options
Item Open Access Contribution of plasma microparticles to the clinical manifestation of allergic diseases(Turkish Society of Immunology, 2012-04) Kahraman, Tamer; Erkoçoğlu, M.; Azkur, D.; Kocabaş, C. N.; Gürsel, İhsanMicroparticles (MPs) are nanovesicles secreted from wide variety of cells. They have role in cellular communications in addition to their physiological roles in several diseases. In this study, we investigated roles of MPs in allergic diseases. Method Peripheral blood from 43 asthma, 15 atopic dermatitis and 13 healthy subjects were collected. MPs were isolated via differential centrifugation and subjected to Annexin-V staining together with different cell specific surface markers (CD9, CD14, CD42a, CD69 and CD105) and analyzed by FACS. Internalization of MPs by PBMCs were assessed by SP-DiOC staining. Results FACS analysis demonstrated that number of MPs in ml plasma was 8,3 ± 1,5 x105, 3,2 ± 1,8 x105, 1,9 ± 0,8 x105, and 3,2 ± 2,2 x105 in healthy, atopic dermatitis, asthma controlled and asthma attack subjects, respectively.Item Open Access CpG loaded flourescent polymeric nanoparticles: a theranostic drug delivery system suitable for TLR based therapies(Turkish Society of Immunology, 2012-04) Kahraman, Tamer; Bayyurt, Banu; İbrahimova, Vusela; Tuncel, Dönüş; Gürsel, İhsanDesigning nanoparticulate delivery systems suitable for simultaneous imaging have been attracting great interest. Several systems such as liposomes, micelles, dendrimers, nanospheres and nanocapsules are potential theranostic carriers in biomedical applications. Here, for the first time we describe a complexation strategy allowing us to deliver a TLR ligand along with simultaneous imaging of the tissues with fluorescent polymeric nanoparticles (NPs). METHOD: Four different NPs (P1, P2, P3 and P4) were used for biocompatibility and drug delivery experiments. RAW264.7 cells were incubated with NPs for indicated time periods and analyzed by FACS and confocal microscopy. Cytotoxicity experiments were performed with Dojindo reagent on RAW264.7 cells. In addition, cellular uptake of NPs in the presence of several scavenger receptor ligands was also investigated. Next, NPs were incubated with CpG ODN to yield nanocomplexes. In vitro and in vivo immunostimulatory effect of nanocomplexes was analyzed both on PBMCs or splenocytes. Pro-inflammatory cytokine production was assessed either by ELISA or in some cases by PCR.Item Open Access CpG ODN loaded exosome nanovesicles: enhanced immunostimulatory activity(Turkish Society of Immunology, 2012-04) Güçlüler, Gözde; Kahraman, Tamer; Gürsel, İhsanExosomes are naturally occurring, membranous nanovesicles known to function as intercellular communication vectors. To explore whether these naturally occuring bilayer vesicles could be exploited as a nucleic acid delivery system we intentionally loaded exosomes with TLR9 ligands and studied their immunostimulatory properties. METHODS: Exosomes were isolated from RAW264.7 and EG-7 cell supernatants by differential centrifugation, filtration and ultracentrifugation and were loaded with CpG ODNs via dehydration-rehydration protocol. Splenocytes were stimulated and analyzed by flow cytometry for cell surface marker upregulation, intracellular cytokine production and co-stimulatory molecule upregulation. Confocal microscopy analyses were done to assess internalization properties of these nanovesciles. Cell supernatants were studied by ELISA to assess Th1 biased cytokine/chemokine secretion.Item Open Access Effects of obesity on airway and systemic inflammation in asthmatic children(S. Karger AG, 2021-03-22) Vezir, Emine; Civelek, Ersoy; Dibek Misirlioglu, Emine; Toyran, Muge; Capanoglu, Murat; Karakus, Esra; Kahraman, Tamer; Ozguner, Meltem; Demirel, Fatma; Gürsel, İhsan; Kocabas, Can NaciObese asthma is a complex syndrome with certain phenotypes that differ in children and adults. There is no clear evidence regarding the presence of additive or synergistic pathological interaction between obesity and asthma in children. Objectives: Our aim was to demonstrate the interaction of obesity and asthma in children in terms of airway and systemic inflammation by a controlled observational study. Methods: Four groups were formed: asthma obese (AO), asthma nonobese (ANO), non-AO (NAO), nonasthma nonobese (NANO). Spirometry test, fractional exhaled nitric oxide (FeNO) test, skin prick test, serum inflammatory biomarkers (C-reactive protein, C3, C4, adiponectin, leptin, resistin, periostin, YKL-40, Type 1, and Type 2 cytokines) were conducted and evaluated in all participants. Sputum inflammatory cells (sputum eosinophils and neutrophils) were evaluated in patients who could produce induced sputum and obesity-asthma interactions were determined. Results: A total of 153 participants aged 6–18 years were included in the study, including the AO group (n = 46), the ANO group (n = 45), the NAO group (n = 30), and the NANO group (n = 32). IL-4 (p < 0.001), IL-5 (p < 0.001), IL-13 (p < 0.001), resistin (p < 0.001), and YKL-40 (p < 0.001) levels were higher in patients with asthma independent of obesity. The lowest adiponectin level was found in the AO group and obesity-asthma interaction was detected (p < 0.001). Sputum eosinophilia (p < 0.01), sputum neutrophilia (p < 0.01), and FeNO levels (p = 0.07) were higher in asthmatic patients independent of obesity. In the group with paucigranulocytic inflammation, resistin and YKL-40 levels were significantly lower than in the group without paucigranulocytic inflammation (p < 0.01). Conclusion: No interaction was found between obesity and asthma in terms of airway inflammation. Interaction between obesity and asthma was shown in terms of adiponectin level and resistin/adiponectin and leptin/adiponectin ratios. It was found that serum YKL-40 and resistin levels could be associated with airway inflammation.Item Open Access Immunostimulatory activity of CpG-ODN is enhanced via encapsulation into nanovesicles(European Molecular Biology Organization, 2012-05) Güçlüler, Gözde; Kahraman, Tamer; Gürsel, İhsanItem Open Access A novel ODN delivery platform: CpG-ODN-loaded exosome nanovesicles(Association for Cancer Immunotherapy, 2012-05) Kahraman, Tamer; Güçlüler, Gözde; Gürsel, İhsanExosomes are naturally occurring, membranous nanovesicles of 40-100 nm in diameter. They arise from the endocytic cellular pathway through three stages: (i) formation of endocytic vesicles ii) multivesicular bodies (MVBs) formation in the cytosol and (iii) fusion of the MVBs with the plasma membrane to release their nanovesicular cargoes (1, 2).Item Open Access The role of bcsE gene in the pathogenicity of Salmonella(Oxford University Press, 2021-07-19) Özdemir, Caner; Akçelik, N.; Özdemir, F. N.; Evcili, İrem; Kahraman, Tamer; Gürsel, İhsan; Akçelik, M.The effects of the bcsE gene and BcsE protein on bacterial physiology and pathogenicity in SalmonellaTyphimurium and Salmonella Group C1 were investigated. It was observed that biofilm and pellicle formation did not occur in the bcsE gene mutants of wild-type strains. Besides, the ‘rdar’ (red, dry, rough) biofilm morphotype in wild-type strains changed significantly in the mutants. In terms of the bcsE gene, the swimming and swarming motility in mutant strains showed a dramatic increase compared to the wild-type strains. The Salmonella bcsE gene was cloned into Escherichia coli BL21, and the his-tagged protein produced in this strain was purified to obtain polyclonal antibodies in BALB/c mice. The antibodies were showed labeled antigen specificity to the BscE protein. As a result of immunization and systemic persistence tests carried out with BALB/c mice, BscE protein was determined to trigger high levels of humoral and cellular responses (Th1 cytokine production, IgG2a/IgG1 > 1). Systemic persistence in the liver and spleen samples decreased by 99.99% and 100% in the bcsE mutant strains. Finally, invasion abilities on HT-29 epithelial cells of wild-type strains were utterly disappeared in their bcsE gene mutant strains.Item Open Access Targeting PI3K/AKT/mTOR pathway identifies differential expression and functional role of IL8 in liver cancer stem cell enrichment(American Association for Cancer Research, 2019) Kahraman, D. C.; Kahraman, Tamer; Çetin-Atalay, R.Activation of the PI3K/Akt/mTOR pathway is an important signaling mechanism involved in the development and the progression of liver cancer stem cell (LCSC) population during acquired Sorafenib resistance in advanced hepatocellular carcinoma (HCC). Therefore, identification of novel therapeutic targets involving this pathway and acting on LCSCs is highly essential. Here, we analyzed the bioactivities and the molecular pathways involved in the action of small-molecule PI3K/Akt/mTOR pathway inhibitors in comparison with Sorafenib, DNA intercalators, and DAPT (CSC inhibitor) on CD133/EpCAM-positive LCSCs. Sorafenib and DNA intercalators lead to the enrichment of LCSCs, whereas Rapamycin and DAPT significantly reduced CD133/EpCAM positivity. Sequential treatment with Rapamycin followed by Sorafenib decreased the ratio of LCSCs as well as their sphere formation capacity, as opposed to Sorafenib alone. Under the stress of the inhibitors, differential expression analysis of 770 cancer pathway genes using network-based systems biology approach singled out IL8 expression association with LCSCs. Furthermore, IL8 secretion and LCSC enrichment ratio was also positively correlated. Following IL8 inhibition with its receptor inhibitor Reparixin or siRNA knockdown, LCSC features of HCC cells were repressed, and sensitivity of cells to Sorafenib increased significantly. Furthermore, inflammatory cytokines (IL8, IL1β, and IL11) were also upregulated upon treatment with HCC-approved kinase inhibitors Sorafenib and Regorafenib. Hence, chemotherapeutic stress alters inflammatory cytokine gene expression in favor of hepatic CSC population survival. Autocrine IL8 signaling is identified as a critical event, and its inhibition provides a promising complimentary therapeutic approach for the prevention of LCSC population enrichment.Item Open Access Therapeutic and diagnostic applications of extracellular vesicles(Bilkent University, 2016-03) Kahraman, TamerExtracellular vesicles (EV), consisting of exosomes and microvesicles, are secreted biological nanovesicles and assumed plethora of physiological functions ranging from transport of cargo, regulating distant cell communication, and altering immune response. Accumulating evidence suggests that extracellular vesicles may participate in disease pathogenesis of inflammatory diseases. Moreover, accumulating evidence suggests that EVs are promising nanocarrier capable of modulating immune response. This thesis aims to harness EVs in immunotherapeutic and diagnostic applications. Behçet’s Disease (BD) activity is manifested with sustained, over exuberant immune activation, yet the underlying mechanisms leading to active BD state is poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 and EVs are elevated in BD plasma. Our data suggested that majority of LL37 is associated with EVs. This association drives plasma EVs to immune cells, enhancing pathologic and sustained immune response, thereby leading to aggravating BD pathology. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. Findings of this study could be of clinical interest in the management of BD, implicating that LL37/EV association as one of the major contributors of BD pathogenesis and might be used as a diagnostic readout to stratify the severity of BD patients. EVs, more specifically exosomes, suggested as new tools for biomedical applications such as drug/vaccine carrier vesicles. However, efforts to engineer cells to express desired cargo in/on these secreted exosomes or induce physical complexation with candidate bioactive agents or even use of membrane-breaching techniques such as electroporation to load exosomes with desirable cargo showed limited in vivo performance. Here we developed a mild and simple technique enabling external loading of any type of desired bioactive molecule within exosomes at high yield. Using this approach, we exploited therapeutic potential of exosomes encapsulating CpG ODN together with a protein antigen as a vaccine cancer for preventive tumor therapy. CpG ODN loaded within exosomes displayed pronounced in-vitro activity as evidenced by up to 6-fold higher IL6 and IL12 secretion from splenocytes as well as increased IFNα secretion from pDCs. Exosomes protected CpG ODN from digestion by DNase-I up to 90%. In order to demonstrate improved in-vivo activity, exosomes co-encapsulating CpG ODN and ovalbumin were tested as a potential vaccine vector against EG-7 thymoma. Animals that received Exo(CpG ODN+OVA) vaccine led to a magnified and persistent Th1-biased antiOVA IgG responses that was sufficient to fully protect mice from EG-7 derived tumor challenge even after 24 weeks post-booster injection as opposed to free vaccine combination. Our results suggest that EVs could be of clinical interest in both prognosis and management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis. In addition, our studies related with exosomes present a platform that opens a new avenue to personalized cell-free therapeutic intervention and could be developed to harbor other therapeutically important molecules ranging from plasmid to mRNA or si/miRNA for more effective therapeutic modality development in the clinic against debilitating diseases ranging from cancer to infectious diseasesItem Open Access Type I IFN-related NETosis in ataxia telangiectasia and Artemis deficiency(Mosby, 2018) Gül, E.; Sayar, E. H.; Güngör, B.; Kara-Eroğlu, Fehime; Sürücü, N.; Keleş, S.; Güner, S. N.; Fındık, S.; Alpdündar, E.; Ayanoğlu, I. C.; Kayaoğlu, B.; Geçkin, B. N.; Şanlı, H. A.; Kahraman, Tamer; Yakıcıer, C.; Müftüoğlu, M.; Oğuz, B.; Ayvaz, D. N. Ç.; Gürsel, İhsan; Özen, S.; Reisli, İ.; Gürsel, M.Background: Pathological inflammatory syndromes of unknown etiology are commonly observed in ataxia telangiectasia (AT) and Artemis deficiency. Similar inflammatory manifestations also exist in patients with STING-associated vasculopathy in infancy (SAVI). Objective: We sought to test the hypothesis that the inflammation-associated manifestations observed in patients with AT and Artemis deficiency stem from increased type I IFN signature leading to neutrophil-mediated pathological damage. Methods: Cytokine/protein signatures were determined by ELISA, cytometric bead array, or quantitative PCR. Stat1 phosphorylation levels were determined by flow cytometry. DNA species accumulating in the cytosol of patients' cells were quantified microscopically and flow cytometrically. Propensity of isolated polymorhonuclear granulocytes to form neutrophil extracellular traps (NETs) was determined using fluorescence microscopy and picogreen assay. Neutrophil reactive oxygen species levels and mitochondrial stress were assayed using fluorogenic probes, microscopy, and flow cytometry. Results: Type I and III IFN signatures were elevated in plasma and peripheral blood cells of patients with AT, Artemis deficiency, and SAVI. Chronic IFN production stemmed from the accumulation of DNA in the cytoplasm of AT and Artemis-deficient cells. Neutrophils isolated from patients spontaneously produced NETs and displayed indicators of oxidative and mitochondrial stress, supportive of their NETotic tendencies. A similar phenomenon was also observed in neutrophils from healthy controls exposed to patient plasma samples or exogeneous IFN-α. Conclusions: Type I IFN-mediated neutrophil activation and NET formation may contribute to inflammatory manifestations observed in patients with AT, Artemis deficiency, and SAVI. Thus, neutrophils represent a promising target to manage inflammatory syndromes in diseases with active type I IFN signature.