Publications by authors named "Utku Horzum"

13 Publications

  • Page 1 of 1

Functional responsiveness of memory T cells from COVID-19 patients.

Cell Immunol 2021 07 17;365:104363. Epub 2021 Apr 17.

Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey. Electronic address:

The presence of memory T cells in COVID-19 patients has been acknowledged, however the functional potency of memory responses is critical for protection. In this study, naïve, effector, effector memory, and central memory CD4 and CD8 T cells obtained from the COVID-19 survivors were re-exposed to autologous monocyte-derived DCs that were loaded with SARS-CoV-2 spike glycoprotein S1. Proliferation capacity, CD25, 4-1BB, and PD-1 expression, and IFN-γ, IL-6, granzyme, granulysin, and FasL secretion were enhanced in CD4 and CD8 effector memory and central memory T cells. Albeit being at heterogeneous levels, the memory T cells from the individuals with COVID-19 history possess functional capacities to reinvigorate anti-viral immunity against SARS-CoV-2.
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http://dx.doi.org/10.1016/j.cellimm.2021.104363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052500PMC
July 2021

Differential expansion of circulating human MDSC subsets in patients with cancer, infection and inflammation.

J Immunother Cancer 2020 09;8(2)

Medical Center, Department of Neurosurgery, Radboud University, Nijmegen, Gelderland, The Netherlands.

Background: Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells.

Methods: We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders.

Results: We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC.

Conclusions: This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation.
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http://dx.doi.org/10.1136/jitc-2020-001223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481096PMC
September 2020

Leptin promotes proliferation of neonatal mouse stem/progenitor spermatogonia.

J Assist Reprod Genet 2020 Nov 25;37(11):2825-2838. Epub 2020 Aug 25.

Department of Histology and Embryology, Hacettepe University Faculty of Medicine, Ankara, Turkey.

Purpose: To keep and increase spermatogonial stem cell number (SSC) is the only available option for pediatric cancer survivors to maintain fertility. Leptin is secreted by the epididymal white adipose tissue and has receptors on stem/progenitor spermatogonia. The purpose of this study is to demonstrate dose- and time-dependent proliferative effect of leptin on stem/progenitor spermatogonia cultures from prepubertal mice testes.

Methods: CD90.2 (+) stem/progenitor spermatogonia were isolated from the C57BL/6 mouse testis on postnatal day 6 and placed in culture. The proliferative effect of leptin supplementation was assessed by colony formation (diameter and number), WST proliferation assays, and xCELLigence real-time cell analysis (RTCA) on days 3, 5, and 7 of culture. Expressions of p-ERK1/2, p-STAT3, total STAT3, and p-SHP2 levels were determined by western blot analysis.

Results: Leptin supplementation of 100 ng/ml increased the diameter (p = 0.001) and number (p = 0.01) of colonies in stem/progenitor spermatogonial cultures and caused higher proliferation by WST-1 (p = 0.009) compared with the control on day 7. The EC50 was calculated as 114 ng/ml for leptin by RTCA. Proliferative dose of leptin induced increased expression of p-ERK1/2 (p = 0.009) and p-STAT3 (p = 0.023) on stem/progenitor spermatogonia when compared with the untreated group.

Conclusion: The results indicated that leptin supplementation exhibited a dose- and time-dependent proliferative effect on stem/progenitor spermatogonia that was associated with increased expression of ERK1/2 and STAT3 pathways while maintaining their undifferentiated state. This output presents a new agent that may help to expand the stem/progenitor spermatogonia pool from the neonatal testis in order to autotransplant after cancer treatment.
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http://dx.doi.org/10.1007/s10815-020-01929-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642194PMC
November 2020

Clinical Relevance of Polymorphonuclear Myeloid-Derived Suppressor Cells in Autoimmune-Blistering Disorders Pemphigus Vulgaris and Bullous Pemphigoid.

J Invest Dermatol 2021 03 10;141(3):672-675.e1. Epub 2020 Aug 10.

Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey.

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http://dx.doi.org/10.1016/j.jid.2020.07.015DOI Listing
March 2021

Human splenic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) are strategically located immune regulatory cells in cancer.

Eur J Immunol 2020 12 2;50(12):2067-2074. Epub 2020 Sep 2.

Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey.

In contrast to the mouse, functional assets of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in the human spleen remain to be better elucidated. Here, we report that the spleen in gastric and pancreatic cancer adopts an immune regulatory character, harbors excessive amount of PMN-MDSC, and anatomically enables their interaction with T cells. Compared to the peripheral blood, the spleen from cancer patients contained significantly higher levels of low-density PMN-MDSC, but not early-stage MDSC (e-MDSC) and monocytic-MDSC (M-MDSC). Low-density fraction of polymorphonuclear (PMN) cells was enriched in immature myeloid cells and displayed higher levels of CD10, CD16, and ROS than their blood-derived counterparts. They were also positive for PD-L1, LOX-1, and pSTAT3. The white pulp and periarteriolar lymphoid sheath (PALS) were strategically surrounded by PMN cells that were in contact with T cells. Unlike those from the blood, both low-density and normal-density PMN cells from the human spleen suppressed T cell proliferation and IFN-γ production. Independent of clinical grade, high PMN-MDSC percentages were associated with decreased survival in gastric cancer. In summary, our results outline the immune regulatory role of the spleen in cancer where neutrophils acquire MDSC functions and feasibly interact with T cells.
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http://dx.doi.org/10.1002/eji.202048666DOI Listing
December 2020

CD66b monocytes represent a proinflammatory myeloid subpopulation in cancer.

Cancer Immunol Immunother 2021 Jan 6;70(1):75-87. Epub 2020 Jul 6.

Department of Basic Oncology, Hacettepe University Cancer Institute, 06230, Ankara, Turkey.

Myeloid-derived suppressor cells (MDSC) populate the peripheral blood and contribute to immune regulation in cancer. However, there is limited knowledge on the myeloid cell types with proinflammatory capacities that may serve as opponents of MDSC. In the circulation of cancer patients, a monocyte subpopulation was identified with a specific immunophenotype and transcriptomic signature. They were predominantly CD14CD33CD16HLA-DR cells that typically expressed CD66b. In accordance with the transcriptomics data, NALP3, LOX-1 and PAI-1 levels were also significantly upregulated. The CD66b monocytes displayed high phagocytic activity, matrix adhesion and migration, and provided costimulation for T cell proliferation and IFN-γ secretion; thus, they did not suppress T cell responses. Irrespective of clinical stage, they were identified in various cancers. In conclusion, the CD66b monocytes represent a novel myeloid subpopulation which is devoid of immune regulatory influences of cancer and displays enhanced proinflammatory capacities.
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http://dx.doi.org/10.1007/s00262-020-02656-yDOI Listing
January 2021

PD-L2 wound zone macrophage-like cells display M1/M2-mixed activation and restrain the effector Th1 responses.

Immunol Cell Biol 2020 02 13;98(2):152-164. Epub 2020 Jan 13.

Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey.

Depending on the microenvironment conditions, macrophages display phenotypic and functional heterogeneity. This study characterized the programmed cell death-ligand 2 (PD-L2)-expressing macrophage-like cells drained from surgical wound zones, and investigated their influence on helper T (Th) cell responses. Although all CD14 myeloid cells possessed macrophage-like features, CD206 and CD163 cells constituted a specific subpopulation with high PD-L2 expression. There was a modest correlation between the PD-L2 levels on CD206 macrophages and the amount of interferon (IFN)-γ in the drainage fluid. The adhesion-independent macrophages simultaneously presented both classically-activated M1 and alternatively-activated M2 characteristics. CD206 and PD-L2 cells were identified with high granularity and size, expressed arginase-1 and costimulatory molecules, had enhanced phagocytic activity and produced reactive oxygen species. The genes associated with macrophage differentiation (MERTK, AXL and TYRO3) were also upregulated. These cells provided costimulation to Th cells; yet, when PD-L2 was blocked, T-cell proliferation and IFNγ production were enhanced. Under defined conditions devoid of activation stimuli and matrix adhesion, ex vivo-generated monocyte-derived macrophages displayed limited capacity to stimulate T cells. Upon exposure to IFNγ, they significantly upregulated programmed death 1 ligands, especially PD-L2. These cells did not completely abrogate T-cell differentiation; however, PD-L2 checkpoint blockade restored Th1 proliferation and secretion of interleukin-2, tumor necrosis factor-α and IFNγ. In conclusion, upregulation of PD-L2 on the wound zone macrophages may constitute a negative feedback loop that restrains the Th1 effector responses and avoids exacerbation of inflammation during tissue healing.
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http://dx.doi.org/10.1111/imcb.12310DOI Listing
February 2020

Myeloid maturation potentiates STAT3-mediated atypical IFN-γ signaling and upregulation of PD-1 ligands in AML and MDS.

Sci Rep 2019 08 12;9(1):11697. Epub 2019 Aug 12.

Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey.

Interferon (IFN)-γ is the major mediator of anti-tumor immune responses; nevertheless, cancer cells use intrigue strategies to alter IFN-γ signaling and avoid elimination. Understanding the immune regulatory mechanisms employed by acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells upon exposure to IFN-γ is critical for development of immunotherapy and checkpoint blockade therapy approaches. This study aims to explore the influence of myeloid maturation on IFN-γ-induced PD-L1 and PD-L2 expression and on pro-leukemogenic transcription factor STAT3 signaling in AML and MDS. Stimulation of myeloid blasts' maturation by all-trans retinoic acid (ATRA) or 1α,25-dihydroxyvitamin D3 (vitamin D) increased the CD11b fraction that expressed PD-1 ligands in response to IFN-γ. Intriguingly, STAT3 pathway was potently induced by IFN-γ and strengthened upon prolonged exposure. Nonetheless, STAT3-mediated atypical IFN-γ signaling appeared as a negligible factor for PD-L1 and PD-L2 expression. These negative influences of IFN-γ could be alleviated by a small-molecule inhibitor of STAT3, stattic, which also inhibited the upregulation of PD-L1. In conclusion, induction of myeloid maturation enhances the responsiveness of AML and MDS cells to IFN-γ. However, these malignant myeloid cells can exploit both STAT3 pathway and PD-1 ligands to survive IFN-γ-mediated immunity and maintain secondary immune resistance.
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http://dx.doi.org/10.1038/s41598-019-48256-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691003PMC
August 2019

Efficacy of a novel LyP-1-containing self-microemulsifying drug delivery system (SMEDDS) for active targeting to breast cancer.

Eur J Pharm Biopharm 2019 Mar 22;136:138-146. Epub 2019 Jan 22.

Department of Pharmaceutical Technology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey. Electronic address:

An ideal cancer therapy targets the tumor cells selectively without damaging healthy tissues. Even though the tumor-specific markers are limited, these molecules can be used for the delivery of anti-cancer drugs as an active targeting strategy. Since the lymphatic system plays a critical role in the dissemination of cancer cells, the drugs directed through lymphatics can feasibly reach to the sites of metastasis. LyP-1 is a peptide that binds to the p32 receptor which is highly expressed not only on the lymphatic endothelium but also on the malignant cells; thus, making this peptide ligand a preferable candidate to mediate active targeting of lymphatics and cancer cells. In this study, different formulations of LyP-1 containing lipid-based nanopharmaceutics so-called self-microemulsifying drug delivery systems (SMEDDS) were developed and tested for their efficacy in targeting breast cancer. Following the selection of non-toxic formulation, doxorubicin hydrochloride and LyP-1 were co-administered in the SMEDDS, which resulted in a significant increase in in vitro cytotoxicity in p32-expressing breast cancer cells, 4T1 and MDA-MB-231. Accordingly, the uptake of LyP-1 in the SMEDDS by the cancer cells was demonstrated. The expression of p32 was detected in the 4T1 tumor tissues which were efficiently targeted with LyP-1 in the SMEDDS. When doxorubicin was co-administrated with LyP-1 in SMEDDS via intraperitonial administration, tumor growth and metastasis were significantly reduced. In conclusion, a novel and efficacious SMEDDS formulation containing LyP-1 with a droplet size less than 100 nm was developed for the lymphatic targeting of breast cancer.
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http://dx.doi.org/10.1016/j.ejpb.2019.01.017DOI Listing
March 2019

Evaluation of brain-targeted chitosan nanoparticles through blood-brain barrier cerebral microvessel endothelial cells.

J Microencapsul 2017 Nov 13;34(7):659-666. Epub 2017 Sep 13.

a Department of Pharmaceutical Technology, Faculty of Pharmacy , Hacettepe University , Ankara , Turkey.

The blood-brain barrier (BBB) is the major problem for the treatment of central nervous system diseases. A previous study from our group showed that the brain-targeted chitosan nanoparticles-loaded with large peptide moieties can rapidly cross the barrier and provide neuroprotection. The present study aims to determine the efficacy of the brain-targeted chitosan nanoparticles' uptake by the human BBB cerebral microvessel endothelial cells (hCMECs) and to investigate the underlying mechanisms for enhanced cellular entry. Fluorescently labelled nanoparticles either conjugated with antibodies recognising human transferrin receptor (anti-TfR mAb) or not were prepared, characterised and their interaction with cerebral endothelial cells was evaluated. The antibody decoration of chitosan nanoparticles significantly increased their entry into hCMEC/D3 cell line. Inhibition of cellular uptake by chlorpromazine indicated that the anti-TfR mAb-conjugated nanoparticles were preferentially cell internalised through receptor-mediated endocytosis pathway. Alternatively, as primarily observed with control chitosan nanoparticles, aggregation of nanoparticles may also have induced macropinocytosis.
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http://dx.doi.org/10.1080/02652048.2017.1375039DOI Listing
November 2017

A small variation in average particle size of PLGA nanoparticles prepared by nanoprecipitation leads to considerable change in nanoparticles' characteristics and efficacy of intracellular delivery.

Artif Cells Nanomed Biotechnol 2017 Dec 13;45(8):1657-1664. Epub 2017 Jan 13.

a Department of Pharmaceutical Technology, Faculty of Pharmacy , Hacettepe University , Ankara , Turkey.

In this study, it was aimed to investigate characteristics and intracellular delivery of two different-sized PLGA nanoparticles in ouzo region by considering number of nanoparticles. To determine the effect of formulation parameters on average particle size, Dil labeled nanoparticles were prepared using a three-factor, two-level full factorial statistical experimental design. PLGA (230.8 ± 4.32 nm) and PLGA (157.9 ± 6.16 nm) nanoparticles were obtained by altering polymer amount based on experimental design results and characterized. Same number of PLGA and PLGA nanoparticles per cell were applied onto HEK293 cells; then, cytotoxicity, uptake kinetics and mechanism were evaluated by flow cytometry and fluorescent microscopy. Also same weight of PLGA and PLGA nanoparticles were applied and cellular uptake of these nanoparticles was evaluated. It was found that PLGA nanoparticles had higher encapsulation efficiency and slower dye release compared to PLGA nanoparticles. When they were applied at same counts per cell, PLGA nanoparticles displayed faster and higher intracellular dye transfer than PLGA nanoparticles. On the other hand, PLGA appeared to be a more effective vehicle than PLGA when applied at the same weight concentration. It was also shown that for both nanoparticles, HEK293 cells employed macropinocytic, caveolae- and clathrin-mediated endocytic pathways.
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http://dx.doi.org/10.1080/21691401.2016.1276924DOI Listing
December 2017

Differentiation of Normal and Cancer Cell Adhesion on Custom Designed Protein Nanopatterns.

Nano Lett 2015 Aug 7;15(8):5393-403. Epub 2015 Jul 7.

Department of Molecular Biology and Genetics, Izmir Institute of Technology, 35430 Urla/Izmir, Turkey.

Cell adhesion to the extracellular matrix is deregulated in metastasis. However, traditional surfaces used to study cell adhesion do not faithfully mimic the in vivo microenvironment. Electron beam lithography (EBL) is able to generate customized protein nanopatterns. Here, we used an EBL-based green lithography approach to fabricate homogeneous and gradient, single (fibronectin, K-casein) and double (fibronectin, laminin) active component protein nanopatterns with micrometer scale spacing to investigate differences in adhesion of breast cancer cells (BCC) and normal mammary epithelial cells (NMEC). Our results showed that as expected, in contrast to NMEC, BCC were plastic: they tolerated nonadhesion promoting regions, adapted to flow and exploited gradients better. In addition, the number of focal adhesions but not their area appeared to be the dominant parameter for regulation of cell adhesion. Our findings also demonstrated that custom designed protein nanopatterns, which can properly mimic the in vivo microenvironment, enable realistic distinction of normal and cancerous cell adhesion.
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http://dx.doi.org/10.1021/acs.nanolett.5b01785DOI Listing
August 2015

Step-by-step quantitative analysis of focal adhesions.

MethodsX 2014 7;1:56-9. Epub 2014 Jul 7.

Izmir Institute of Technology, Department of Molecular Biology and Genetics, 35430 Izmir, Turkey.

Focal adhesions (FAs) are specialized adhesive structures which serve as cellular communication units between cells and the surrounding extracellular matrix. FAs are involved in signal transduction and actin cytoskeleton organization. FAs mediate cell adhesion, which is a critical phenomenon in cancer research. Since cells can form many and micrometer scale FAs, their quantitative analysis demands well-optimized image analysis approaches [1-3]. Here, we have optimized the analysis of FAs of MDA-MB-231 breast cancer cells. The optimization is based on proper processing of immunofluorescence images of vinculin, which is one of the markers of FAs. All image processing steps are carried out using the ImageJ software, which is freely available and in the public domain. The advantages of our method are:•The analysis steps are simplified by combining different plugins of the ImageJ program.•FAs are better detected with minimal false negatives due to optimized processing of fluorescent images.•This approach can be applied to quantify a variety of fluorescent images comprising focal and/or localized signals within a high background such as FAs, one of the many complex signaling structures in a cell.
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http://dx.doi.org/10.1016/j.mex.2014.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472847PMC
July 2015
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