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Öğe A Study of the Effects of Metformin, a Biguanide Derivative, on Annulus Fibrosus and Nucleus Pulposus Cells(Turkish Neurosurgical Soc, 2020) Kaya, Yasin Emre; Karaarslan, Numan; Yılmaz, İbrahim; Şirin, Duygu Yaşar; Akalan, Hande; Özbek, HanefiAIM: To investigate the effects of metformin, a drug used widely for the treatment of type 2 diabetes mellitus, on human primary cell cultures prepared from uninjured segment of disc material intervertebral disk tissues. MATERIAL and METHODS: Primary cell cultures were prepared using the tissues of six patients (three males and three females) who had undergone lumbar microdiscectomy and sequestrectomy. Untreated samples served as the control group, and metformintreated samples served as the experimental group. All the samples were evaluated using an inverted light microscope, acridine orange/propidium iodide staining (AO/PI), and a fluorescence microscope. The cytostatic and cytotoxic effects of metformin, which was administered to the samples using a commercial MTT assay kit, were also evaluated. The data obtained were statistically assessed, and the alpha significance value was accepted as less than 0.05. In addition, for the groups' changes in the expressions of chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1 beta (IL-1 beta) matrix metalloproteinase 7 (MMP-7), and matrix metalloproteinase 19 (MMP-19), genes related to the extracellular matrix synthesis and degradation were determined using gene-specific TaqMan Gene Expression Assays. RESULTS: The administration of the drug adversely affected nucleus pulposus (NP)/annulus fibrosus (AF) cells and extracellular matrix-like structures. This was statistically significant (p<0.05). CONCLUSION: Clinicians should not disregard the adverse effects of metformin, which is used widely in clinical practice, on the components of intervertebral disk tissues.Öğe A Study on the Effects of Direct Factor Xa Inhibitors and Direct Thrombin Inhibitors on Human Primary Chondrocyte Cultures(2019) Kaya, Yasin Emre; Akalan, Hande; Yılmaz, İbrahim; Karaarslan, Numan; Şirin, Duygu Yaşar; Özbek, Hanefi; Ateş, ÖzkanAim:This study investigates the effects of two direct factor Xa inhibitors, apixaban and rivaroxaban, and a direct thrombin inhibitor, dabigatran, on human primary chondrocytecultures.Materials and Methods:Monolayer cultured chondrocytes were prepared. Cell cultures were treated with dabigatran, apixaban, and rivaroxaban. Cultures without drug treatments served as the control group. Using an inverted light microscope, the cell surface morphology was examined. Cell viability and the toxicity of drugs were evaluated using a commercial assay kit, and the results were confirmed using two nucleic acid binding dyes, acridine orange and propidium iodide. The expressions of cartilage oligomeric protein, matrix metalloproteinase-7, and matrix metalloproteinase-19 were assessed using the real-time polymerase chain reaction analysis. All the analyses were performed within 21 days. The data obtained were statistically evaluated.Results:The administration of the three drugs changed the cell viability, proliferation, and expressions of cartilage oligomeric protein, matrix metalloproteinase-7, and matrix metalloproteinase-19. The results were statistically significant (P<0.05).Conclusion:Results obtained from in vitro studies may not provide accurate and reliable insight for clinical practices. However, clinicians should know that drugs used for the prevention or treatment of diseases may suppress chondrocyte proliferation and damage the extracellular matrix formation.Öğe Alloreactive memory B cell detection by flow cytometric cross match using polyclonally activated memory B cell culture supernatants(Elsevier, 2022) Akalan, Hande; Şirin, Duygu Yaşar; Yılmaz, İpek; Ata, Pınar; Kara, Veli Melih; Taşdemir, Nicel; Bilgen, Türker; Titiz, Mesut İzzetIn addition to alloantibodies, alloreactive memory B cell (mBC) evaluation has a potential for immunological risk assessment during transplantation processes. For the alloreactive mBCs evaluation currently, direct Flow Cytometric (FC) analysis using the HLA tetramer staining is an option. Evaluation of alloantibodies produced by the polyclonally stimulated alloreactive mBCs in in vitro culture system seems to be another useful approach, but this needs further downstream applications. In this study, we investigated the usefulness of the Flow Cytometric Cross Match (FCXM-supernatant) in which in vitro polyclonally activated mBCs culture supernatants and potential donor's lymphocytes being used for the mBC detection. FCXM-supernatant assays were performed between culture supernatants of polyclonally activated mBCs obtained from 4 allosensitized multiparous women and 14 renal transplant patients, and their non-alloimmunized spouses' or donors' lymphocytes, and vice versa. HLA typing was performed by SSP method. Anti-HLA antibodies produced by in vitro activated alloreactive mBCs were also evaluated by the Luminex assays. The success of in vitro polyclonal activation of mBCs was evaluated by a total IgG ELISA test and antibody secreting cell analyses by FC. Donor specific alloreactive mBCs were detected by FCXM-supernatant in 45% of the 18 allosensitized cases. Detection rate was 85% (6 out of 7) in the strongly allosensitized cases. No alloreactive mBCs was detected in control cases without allosensitization. FCXMsupernatant negative results of the allosensitized cases were related to low level of allosensitization and insufficient polyclonal stimulation evaluated by total IgG antibody tests of the supernatants. We herein report a practical methodology for alloreactive mBC detection as a donor specific manner using the FCXM-supernatant assay so that this would easily be transformed into a routine test performed in tissue typing laboratories.Öğe Can transcription factors in the intervertebral disc of lopinavir/ritonavir prevent degeneration in the nucleus pulposus by mediating the regulation of inflammation through signaling pathways?(Verduci Editore s.r.l, 2022) Yılmaz, İbrahim; Akalan, Hande; Karaarslan, Numan; Şirin, Duygu Yaşar; Kaplan, Nuray; Doğan, Mustafa; Ateş, OğuzOBJECTIVE: This study was conducted to examine whether lopinavir/ritonavir (Lop/r), an HIV protease inhibitor, can improve disc physiology and slow down intervertebral disc (IVD) degeneration through in vitro experimental methods, as well as whether it can suppress inflammation with interleukin-1 beta (IL-1?) and sex-determining region Y (SRY) protein-related high-mobility group box genes-9 (SOX9) through hypoxia-inducible factor 1-alpha (HIF-1?) and the nuclear factor kappa B (NF-?B) signaling pathway. The aim was to investigate whether Lop/r application is toxic to IVD cells and the microenvironment simultaneously. PATIENTS AND METHODS: Human primary cell cultures were prepared using herniated IVD tissues obtained from patients with lumbar disc hernia who were unresponsive to conservative and medical treatment, and thereby, were operated on. The untreated culture samples served as control group, and the samples treated with Lop/r served as study group. Microscopic evaluations were performed simultaneously using fluorescent and supravital dyes in all groups. In addition to cell viability, toxicity, and proliferation analysis through a commercial kit, IL-1?, SOX9, HIF-1?, and NF-?B protein expressions were evaluated using Western blotting. In the statistical comparison of the obtained data, an alpha value less than 0.05 was considered significant. RESULTS: Cell proliferation decreased in the Lop/r group, but no cell death was observed (p < 0.05). Moreover, at the end of 72 hours after Lop/r application, IL-1? and NF-kB protein expressions decreased by 40% and 52%, respectively, while HIF-1? and SOX9 protein expressions increased by 4% and 59%, respectively (p < 0.05). CONCLUSIONS: Although these data were obtained from an in vitro experimental study, it is believed that these findings could make significant contributions to the pharmaco-regenerative treatment modalities of IVD degeneration. Lop/r suppresses the IL-1? and NF-?B and induces SOX9 and HIF-1?, since these signaling pathways may be related to human IVD degeneration. © 2022 Verduci Editore s.r.l. All rights reserved.Öğe Can transcription factors in the intervertebral disc of lopinavir/ritonavir prevent degeneration in the nucleus pulposus by mediating the regulation of inflammation through signaling pathways?(Verduci Publisher, 2022) Yılmaz, I.; Akalan, Hande; Karaarslan, N.; Şirin, Duygu Yaşar; Kaplan, N.; Doğan, Mustafa; Özbek, H.OBJECTIVE: This study was con-ducted to examine whether lopinavir/ritonavir (Lop/r), an HIV protease inhibitor, can improve disc physiology and slow down intervertebral disc (IVD) degeneration through in vitro experimental methods, as well as whether it can suppress inflammation with interleukin-1 beta (IL-113) and sex-determining region Y (SRY) protein-related high-mobility group box genes-9 (SOX9) through hypoxia-inducible factor 1-alpha (HIF-1 alpha) and the nuclear factor kappa B (NF-KB) signaling pathway. The aim was to investigate whether Lop/r application is toxic to IVD cells and the microenvironment simultaneously. PATIENTS AND METHODS: Human primary cell cultures were prepared using herniated IVD tissues obtained from patients with lumbar disc hernia who were unresponsive to conservative and medical treatment, and thereby, were operated on. The untreated culture samples served as control group, and the samples treated with Lop/r served as study group. Microscopic evaluations were performed simultaneously using fluorescent and supravital dyes in all groups. In addition to cell viability, toxicity, and proliferation analysis through a commercial kit, IL-113, SOX9, HIF-1 alpha, and NF-KB protein expressions were evaluated using Western blotting. In the statistical comparison of the obtained data, an alpha value less than 0.05 was considered significant. RESULTS: Cell proliferation decreased in the Lop/r group, but no cell death was observed (p < 0.05). Moreover, at the end of 72 hours after Lop/r application, IL-113 and NF-kB protein expressions decreased by 40% and 52%, respectively, while HIF-1 alpha and SOX9 protein expressions increased by 4% and 59%, respectively (p < 0.05). CONCLUSIONS: Although these data were obtained from an in vitro experimental study, it is believed that these findings could make significant contributions to the pharmaco-regenerative treatment modalities of IVD degeneration. Lop/r suppresses the IL-113 and NF-KB and induces SOX9 and HIF-1 alpha, since these signaling path-ways may be related to human IVD degeneration.Öğe Does oseltamivir protect human chondrocyte and nucleus pulposus cells from degeneration by inhibiting senescence and proinflammation mediated by the NLRP3 inflammasome and NF-kappa B?(Verduci Publisher, 2022) Yılmaz, İbrahim; Akalan, Hande; Öznam, K.; Karaarslan, Numan; Şirin, Duygu Yaşar; Özbek, HanefiOBJECTIVE: Recent drug design studies suggest that inflammation is among the most important factors in the development of both intervertebral disc (IVD) degeneration (IVDD) and osteoarthritis (OA) due to cartilage damage. This study aimed to investigate whether the anti-inflammatory drug oseltamivir has a toxic effect on IVD and cartilage tissue cells. It assessed what effect oseltamivir has on hypoxia-inducible factor (HIF)-1 alpha (HIF1 alpha), which plays an important role in anabolic pathways in IVD and cartilage tissue. In addition, the study analyzed whether oseltamivir could inhibit the release of inflammatory interleukin-1 beta (IL-1 beta) via the nuclear factor kappa-B (NF-kappa B) signaling pathway by activating the nucleotide-binding oligomerization domain and leucine-rich repeat protein-3 (NLRP3) inflammasome. MATERIALS AND METHODS: Human lumbar IVD (n = 8) tissues were isolated for annulus fibrosus (AF) and nucleus pulposus (NP) primary cell cultures. and human tibial and femoral cartilage tissues (n = 8) were isolated for primary chondrocyte cultures. Untreated groups served as the control and oseltamivir-treated groups as the study sample. Cell viability and cytotoxicity were evaluated at 0, 24, 48. and 72 h in all groups for changes in HIF-1 alpha, IL-18, NF-kappa B. and the NLRP3-inflammasome protein expressions using Western blotting. The a significance value was < 0.05. RESULTS: In the oseltamivir-treated groups, cell proliferation decreased in both AF/NP cell and chondrocyte cultures obtained from IVD cartilage tissues. After Western blotting analysis, changes were observed in the protein expressions of HIF-1 alpha, IL-1 beta, NF-kappa B, and the NLRP3 inflammasome in both AF/NP cells and chondrocytes. The results were statistically significant (p < 0.05). CONCLUSIONS: Oseltamivir treatment may be a promising regenerative strategy to manage IVDD and osteoarthritic cartilage tissues.Öğe Effects of an acetylcholinesterase inhibitor and an N-methyl-D-aspartate receptor antagonist on inflammation and degeneration of the nucleus pulposus(Verduci Publisher, 2022) Yılmaz, İbrahim; Akalan, Hande; Şirin, Duygu Yaşar; Karaarslan, Numan; Kaplan, Nuray; Özbek, HanefiOBJECTIVE: The study aimed to examine the effects of two drugs, an acetylcholinesterase inhibitor (AChEI) and an N-methyl-D-aspartate receptor (NMDAR) antagonist, on degenerated annulus fibrosus (AF) and nucleus pulposus (NP) cells and the extracellular matrix (ECM) structure in vitro. PATIENTS AND METHODS: Tissue samples were obtained from patients with intervertebral disc herniation (four males and four females; classified as Pfirmann stage IV) and used to prepare cell cultures. Untreated cell culture samples served as the control group. Study group samples were treated with donepezil, memantine or a combination of the two drugs. Cell viability, toxicity and proliferation were evaluated in all groups. Western blotting was used to examine changes in protein expression of signal transducer and activator of transcription 3 (STAT3), phospho-STAT3 (ser727), hypoxia-inducible factor (HIF)-1 alpha (HIF-1 alpha) and nucleotide-binding oligomerisation domain (NOD) leucine-rich repeat (LRR)-containing proteins (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. The alpha significance value was < 0.05. RESULTS: Analysis of the microscopy and commercial kit results revealed that cell proliferation was suppressed. and no cell death was observed. The protein expression levels of NLRP3, STAT3, ser727 and HIF-1 alpha were lower in the samples treated with donepezil and memantine at 72 h (p < 0.05). The protein expression levels of NLRP3, STAT3, ser727 and HIF-1 alpha were higher in the samples treated with the combination of donepezil and memantine (p < 0.05). CONCLUSIONS: The combined administration of memantine a NMDAR antagonist which can prevent neurodegeneration and donepezil an AChEI used for pain relief increased the protein expression levels in the anabolic pathway. However, it did not reduce the protein expression levels in the catabolic pathway. Therefore, further studies are needed to provide extensive insight into whether it may be among the potential targets for the therapy of intervertebral disc (IVD) diseases.Öğe Effects of Tocilizumab on Intervertebral Disc Degeneration, Cell Senescence and Inflammation via BMP-2, Hif-1?, IL-1? and SOX9(Asian Network Scientific Information-Ansinet, 2023) Yilmaz, Ibrahim; Akalan, Hande; Sirin, Duygu Yasar; Karaarslan, Numan; Kasim, Emin; Ozbek, Hanefi; Ates, OzkanBackground and Objective: Immunosuppressive tocilizumab (TCZ), which is frequently used in the treatment of rheumatoid arthritis, can have many side effects as well as an uncontrolled inflammatory response. This study aimed to evaluate the effect of tocilizumab (TCZ) administered to intervertebral disc (IVD) tissues in vitro on the proinflammatory cytokines and proteins of degeneration, senescence and inflammation-related signaling pathways at the pharmaco-molecular level. Materials and Methods: Primary cell cultures were prepared using human IVD tissues obtained during lumbar microdiscectomy. Untreated groups served as the control and TCZ-treated groups as the study sample. Analyses were performed using a commercial kit, supravital and fluorescent dyes. Changes in bone morphogenetic protein (BMP)-2, hypoxia-inducible factor (Hif)1-alpha (Hif1-alpha), interleukin (IL)-1 beta (IL-1 beta) and sex-determining region Y (SRY)-box 9 (SOX9) protein expressions were evaluated using western blotting. An alpha value of less than 0.05 was considered significant. Results: Proliferation decreased in the samples treated with TCZ (10 mu g mLG1 ) on day 15 (p<0.05). Protein expressions of BMP-2, Hif-1 alpha, IL-1 beta and SOX9, which play a vital role in anabolic and catabolic pathways, changed in samples treated with TCZ (10 mu g mLG1 ). Conclusion: This change was statistically significant (p<0.05). Therefore, results concluded that the inflammation, extracellular matrix degradation and nucleus pulposus degeneration after disc herniation are controlled by BMP-2, Hif-1 alpha, IL-1 beta and SOX9.Öğe Evaluation of the expression and proliferation of degenerative markers in primary cell cultures obtained from human intervertebral disc tissue(2020) Kaya, Yasin Emre; Akalan, Hande; Yılmaz, İbrahi?m; Karaarslan, Numan; Şirin, Duygu Yaşar; Özbek, HanefiAim: A major cause of low back pain is disc degeneration. Nevertheless, no specific and reliable markers of the degeneration of thenucleus pulposus (NP) are available. This presented study aimed to examine changes in the expressions of genes in primary cellcultures isolated from intact and degenerated tissues to give insights into the biopathogenesis of intervertebral disc (IVD) tissue.Material and Methods: Tissues of eight patients (n = 8; average age: 41.74 ± 9.86 years) were resected through microdiscectomy,and primary cell cultures were prepared using degenerated disc tissue. The cultured degenerated tissues served as the study group.The samples in the control group comprised the intact tissues of patients (n = 8; average age: 38.68 ± 7.91 years) resected followinga trauma. Morphology of the cell surface were evaluated using an inverted light/fluorescent microscopy at 0 and 24 h on days 10and 21. The expressions of the chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1 beta (IL-1 beta),and matrix metalloproteinase (MMP)-7 and MMP-19 genes were evaluated using the reverse transcription-quantitative polymerasechain reaction (RT-qPCR). The data obtained were statistically analyzed.Results: The four genes investigated, except COMP (P > 0.05), changed significantly in primary cell cultures isolated from degenerativeIVD tissues. This result was statistically significant (P < 0.05). The gene expressions in the samples derived from intact IVD tissueschanged markedly and these changes were associated with proliferation (P < 0.05).Conclusion: Analyzing the changes in gene expression levels associated with IVD should contribute to future studies on theprevention and treatment of such pathologies. The data obtained from the present study will shed light on cellular-based personaltargeted therapies through which genetic information can be transmitted to cells.Öğe Expression Profile of Transcription Factor ELK-1 and ELK-1 Target Genes on Lymphoma-Leukemia Cell Lines(2018) Akalan, Hande; Şirin, Duygu YaşarPrognostic molecular markers identified in leukemia are becomingincreasingly important especially in risk stratification and to determine therapy. Inthis study, we investigate the role of ELK-1 transcription factor and its potentialtarget genes in four cell lines; Daudi, Jurkat, K-562 and HL-60. To evaluate ELK-1,MCPIP, MCL-1, BCL-10, CEBPB and SRF genes expression profiles we haveperformed a Real-time PCR analysis on Daudi, Jurkat, K-562 and HL-60 cell lines.ELK-1 over expression concomitant with SRF overexpression was detected only inDaudi cell line while only SRF overexpression was detected in jurkat cells.Expression of MCPIP, MCL-1, BCL-10 and CEBPB genes were decreased in all celllines. Protein levels or phosphorylation status of ELK-1, BCL-10, CEBPB, MCL-1,MCPIP and SRF, moreover, changes that may occur when ELK-1 continuousoverexpression is provided or completely silenced in these cell lines have not beenevaluated. These questions are suggestions for future investigations.Öğe Investigation of the Effects of Piperlongumine and Doxorubicin Combined Treatment on Cell Death via PTEN in HeLa Cells(2024) Güzelel, Gonca; Akalan, Hande; Bilgen, Türker; Şirin, Duygu YaşarDoxorubicin (Dox), which is used in treating many types of cancer including cervix cancer nevertheless, its effect alone is low especially in recurrent cases. Therefore, investigating of agents that can increase the impact of Dox continues. The aim of the present study is to answer the question: Can Piperlongumine (PL) a natural alkaloid cause an increase in the efficacy of Dox in the HeLa cell line? In this study, the effects of Dox and PL on cell viability by MTT and Acridine orange/propidium iodide staining, and expression levels of the PTEN (Phosphatase and tensin homolog 10) gene by Real-Time PCR and Western-Blot were evaluated in HeLa cells. It was determined that PL combined with Dox increased cell death and suppressed cell proliferation. The PTEN gene expression was decreased in all experimental groups, but the PTEN protein phosphorylation increased in cultures treated with PL and when Dox/PL was combined. The fact that PL application increases the activation of PTEN, which is a tumor suppressor. This indicates that it can be used to increase the effectiveness of Dox in the treatment.Öğe Is Favipiravir a Potential Therapeutic Agent in the Treatment of Intervertebral Disc Degeneration by Suppressing Autophagy and Apoptosis?(Turkish Neurosurgical Soc, 2022) Yılmaz, İbrahim; Akalan, Hande; Şirin, Duygu Yaşar; Karaarslan, Numan; Özbek, Hanefi; Ateş, ÖzkanAIM: To evaluate the effects of favipiravir (FVP) on cell viability and cytotoxicity in human degenerated primary intervertebral disc (IVD) tissue cell cultures. Furthermore, the protein expressions of hypoxia-inducible factor 1 alpha (HIF-1 alpha), nuclear factor-kappa-b (NF-kappa B), and interleukin-1 beta (IL-1 beta) were also examined. MATERIAL and METHODS: Untreated cell cultures served as the control group, named group 1. Cell cultures treated with FVP served as the study group, named group 2. Pharmacomolecular analyses were performed in all groups at 0, 24, 48, and 72 hours (h). Obtained data were evaluated statistically. RESULTS: Cell proliferation was suppressed in the FVP-treated samples compared to the control group samples at 24 and 72 h, and this was statistically significant (p<0.05). Decreased or increased protein expression levels of HIF-1 alpha, NF-kappa B, and IL-1 beta in FVP-treated samples may be an indication of suppression in anabolic events as well as proliferation in IVD cultures. FVP administration showed that AF/NP cells in a culture medium may induce a strong inflammatory response to FVP. This strong inflammatory response is likely to cause slowed proliferation. It may also be a trigger for many catabolic events. NF-kappa B expression increased within the first 24 h and then decreased rapidly. Based on the data obtained, it may be suggested that the rapidly increasing NF-kB may have stimulated the expression of many antiproliferative genes. CONCLUSION: The suppression of IL-1 beta and NF-kB protein expressions in IVD cells treated with FVP is important in the treatment of IVD degeneration (IDD). If the protein expression of HIF-1 alpha could be increased along with the suppression of IL-1 beta and NF-kB, FVP would perhaps be a promising pharmacological agent in the treatment of IDD.Öğe Lösemi hücre hatlarında elk-1 geni ve bu transkripsiyon faktörünün hedefindeki genlerin ekspresyonlarındaki değişimlerin belirlenmesi(Namık Kemal Üniversitesi, 2015) Akalan, HandeAkut lösemi klinik ve biyolojik heterojenite gösteren malign ve klonal bir hematopoetik kök kücre hastalığıdır. Ülkemizde her yıl binlerce insanda görülen löseminin hücresel kökeninin ve hücrelerin malignant değişimini tetikleyen genetik mekanizmaların anlaşılması için özellikle moleküler genetik çalışmalara ihtiyaç duyulmaktadır. Transkripsiyon faktörü olarak fonksiyon gören ELK-1 proteininin miyojenik farklılaşmadaki rolü iyi bilinmektedir. Ancak yaptığımız literatür taramasında ELK-1 aşırı ekspresyonu ile hematolojik malignansiler arasında ilişki bildiren bir yayına rastlanmamıştır. Yaptığımız çalışmada HL-60, Jurkat, Daudi ve K-562 lösemi hücre hatlarında ELK-1’in gen ekspresyonundaki değişimlerin belirlenmesi planlanmıştır. Ayrıca ELK-1’in lökomogenezdeki rolünün aydınlatılabilmesi için beraber fonksiyon gördüğü düşünülen diğer genlerin (BCL10, CEBPB, MCL1, ZC3H12A, SRF) ekspresyon düzeyleri de araştırılmıştır. Proje kapsamında Elk1 geni ve kodladığı transkripsiyon faktörünün hedefinde olan beş gen bölgesinin ekspresyon düzeyleri gerçek zamanlı polimeraz zincir reaksiyonu yöntemiyle karşılaştırılmıştır. Daudi hücre hattında ELK-1 ve SRF gen ifadesinin arttığı, Jurkat hücre hattında ise sadece ELK-1 gen ifadesinin arttığı belirlenmiştir. Diğer hücre hatlarında ELK-1 veya hedefi olabilecek genlerin ifadesinde değişim belirlenmemiştir.Öğe Protective effects of Acetobacter ghanensis against gliadin toxicity in intestinal epithelial cells with immunoregulatory and gluten-digestive properties(Springer Heidelberg, 2022) Doğuer, Çaglar; Akalan, Hande; Demirok, Nazan Tokatlı; Erdal, Berna; Mete, Rafet; Bilgen, TürkerPurpose The aim of this study was to establish whether Acetobacter ghanensis, the probiotic characteristics of which were evaluated previously, attenuates gliadin-induced toxicity in intestinal epithelial cells with gluten-digestive and immunoregulatory properties. Methods A co-culture model of human intestinal epithelial cell (Caco-2) monolayers on top of peripheral blood mononuclear cells (PBMCs) obtained from patients with celiac disease (CD) was established. The gluten-digestive properties of A. ghanensis were determined by checking bacterial growth in a medium containing gluten as the main nitrogen source. The mRNA levels of genes encoding TJ-associated proteins were measured by quantitative real-time PCR (qRT-PCR). The concentrations of IL-6 and TNFac were determined by enzyme-linked immunosorbent assay (ELISA). Results We found that PT-gliadin disrupted intestinal barrier integrity by modulating the expression of TJ-associated genes encoding zonulin (increased by similar to 60%), zonula occludens-1 (ZO-1) (decreased by similar to 22%), and occludin (decreased by similar to 28%) in Caco-2 cells. Furthermore, PT-gliadin treatment in Caco-2 cells was associated with increased concentrations of IL-6 (similar to 1.6-fold) and TNFac (similar to twofold) from PBMCs. These modulatory effects of PT-gliadin, however, were suppressed when Caco-2 cells were subjected to A. ghanensis in the presence of PT-gliadin. As a factor underlying these protective effects, we showed that A. ghanensis could digest gluten peptides. Conclusions To our knowledge, the current study is the first to demonstrate that A. ghanensis improves intestinal barrier functions by attenuating the modulatory effects of PT-gliadin with immunoregulatory and gluten-digestive properties.Öğe The effects of rivaroxaban, an oral anticoagulant, on human IVD primary cultures(Termedia Publishing House Ltd, 2022) Çalışkan, Tezcan; Akalan, Hande; Yılmaz, İbrahim; Karaarslan, Numan; Şirin, Duygu Yaşar; Özbek, HanefiIntroduction: The present study aimed to investigate the potential effects of rivaroxaban, an oral anticoagulant that inhibits the effects of factor Xa, on intact intervertebral disc tissue cells and the extracellular matrix (ECM). Material and methods: Rivaroxaban was applied to primary human cell cultures prepared from tissues of the intervertebral disc. Comparative molecular analyses were performed on non-drug-treated control group samples. Descriptive statistics were presented as the mean +/- standard deviation. An analysis of variance test was performed to determine whether there were significant differences in the mean across the groups. When differences across groups were observed, Tukey's honestly significant difference post-hoc test was used for multiple pairwise comparisons. The significance of the obtained data was determined statistically. The alpha significance value was < 0.05. Results: The cells in the control group and in the rivaroxaban-treated group were viable, healthy, and proliferated (p < 0.05). However, the expression levels of the chondroadherin gene (CHAD), cartilage oligo matrix protein (COMP), matrix metalloproteinase (MMP)-13, and MMP-19 genes were changed (p < 0.05). Conclusions: Although rivaroxaban does not suppress cell proliferation due to morphological, biological, and biochemical changes in the intervertebral disc tissue, it may change the expression of genes that are related to ECM maintenance.Öğe Transplantasyon başarisinda etkili olan donöre özgün alloreaktif hafiza B hücre rezervinin değerlendirilmesi(Tekirdağ Namık Kemal Üniversitesi, 2023) Akalan, Hande; Şirin, Duygu YaşarAlloantikorlara ek olarak, alloreaktif hafıza B hücre (mBC) rezervi, transplantasyon süreçleri sırasında immünolojik risk değerlendirmesi için bir potansiyele sahiptir. Şu anda alloreaktif mBC değerlendirmesi için, HLA tetramer boyama kullanılarak doğrudan Akış Sitometrik (FC) analizi bir seçenektir. Poliklonal olarak uyarılmış alloreaktif mBC'ler tarafından üretilen alloantikorların in vitro kültür sisteminde değerlendirilmesi, başka bir yararlı yaklaşım gibi görünmektedir, ancak bunun daha fazla ek uygulaya ihtiyacı vardır. Bu çalışmada, mBC tespiti için in vitro poliklonal olarak aktive edilmiş mBC kültür süpernatanlarının ve potansiyel donörün lenfositlerinin kullanıldığı Akış Sitometrik Çapraz Eşleştirmenin (FCXM-süpernatant) yararlılığını araştırdık. Allosensitize edilmiş 10 böbrek nakli hastasından elde edilen poliklonal olarak aktive edilmiş mBC'lerin kültür süpernatanları ile bunların alloimmünize edilmemiş donörlerinin lenfositleri ve tam tersi olacak şekilde FCXM-süpernatant deneyleri yapıldı. HLA tiplemesi SSP yöntemi ile yapıldı. İn vitro aktifleştirilmiş alloreaktif mBC'ler tarafından üretilen anti-HLA antikorları da Luminex testleri ile değerlendirildi. mBC'lerin in vitro poliklonal aktivasyonunun başarısı, toplam IgG ELISA testi ve FC tarafından antikor salgılayan hücre analizleri ile değerlendirildi. Donöre özgü alloreaktif mBC'ler, allosensitize edilmiş 10 vakanın %45'inde FCXM-süpernatant tarafından tespit edildi. Güçlü allosensitize vakalarda tespit oranı %85 (7'de 6) idi. Allosensitizasyon olmayan kontrol vakalarında hiçbir alloreaktif mBC tespit edilmedi. Allosensitize vakaların FCXM süpernatant negatif sonuçları, süpernatanların toplam IgG antikor testleri ile değerlendirilen düşük düzeyde allosensitizasyon ve yetersiz poliklonal stimülasyon ile ilişkiliydi. Bu çalışmada, doku tipleme laboratuvarlarında gerçekleştirilen rutin bir teste kolayca dönüştürülebilmesi için FCXM-süpernatant testi kullanılarak donöre özel bir şekilde alloreaktif mBC tespiti için pratik bir metodoloji sunuyoruz.
