Charles University
Faculty of Medicine in Hradec Králové
17
thINTERNATIONAL MEDICAL POSTGRADUATE CONFERENCE
New Frontiers in the Research of Ph.D. Students
Conference of Medical Schools
November 19 - 20, 2020
Organized by
Charles University, Faculty of Medicine in Hradec Králové
Supported by
Specific Research Projekt no. 260546
Under the Auspices of His Magnificence Rector of the Charles University
Tomáš Zima
Hradec Králové
Educational Centre of the Faculty of Medicine
Table of content
General Information ...5
Scientific Programme ... 6
Evaluation Committee ... 8
Presentations ... 9
Author’s Index ... 96
GENERAL INFORMATION
Venue:
Due to the Pandemic situation COVID-19 the Conference takes place in an offline form
Official Language:
English
Organizing Committee
Miroslav Červinka Jiří Manďák
Editor:
Miroslav Červinka
Technical Assistance:
Petra Malá
The publication has undergone neither linguistic editing nor proof reading. It is printed from the author’s e-mail correspondence.
SCIENTIFIC PROGRAMME
THURSDAY AND FRIDAY NOVEMBER 19 - 20
1. Bavlovič Jan (Hradec Králové, Czech Republic): Study of the Secretion of Outer Membrane Vesicles in the bacterium F. Tularensis and their Role in the Host- Pathogen Interaction
2. Bolek Tomáš (Martin, Slovak Republic): The Impact of Proton Pump Inhibitors on Direct Oral Anticoagulants Activity in Patients with Atrial Fibrillation
3. Csabai Tímea (Pécs, Hungary): Altered Immune Response and Implantation Failure in Progesterone-Induced Blocking Factor-Deficient Mice
4. Daniel Petr (Prague, Czech Republic): Differentially Expressed Mitochondrial Proteins in Multi-Step Selected Taxane-Resistant MCF-7 Breast Cancer Cells 5. Fell Christopher (Vienna, Austria): A Novel Role for FIBCD1 in the Brain 6. Ferencová Nikola (Martin, Slovak Republic): Sympathetic Nervous System
Activity in Autism Spectrum Disorder – Effect of Posture and Treatment 7. Koutová Darja (Hradec Králové, Czech Republic): Montanine-Type
Amaryllidaceae Alkaloid Pancracine: Mechanistic Insight into Antiproliferative and Cytotoxic Activity
8. Kroupová Anna (Prague, Czech Republic): Effect of Perinatal Stress and Drug Abuse on Anxiety-Like Behavior of Adult Male Rats in Open Field and Elevated Plus Maze
9. Naylor-Adamson Leigh (Hull, United Kingdom): BTK Inhibitors Ibrutinib and Acalabrutinib Impair FCγRIIA-Mediated Responses of Healthy Donor Platelets and Chronic Lymphocytic Leukaemia Platelets to Bacteria
10. Nita Alexandru (Brno, Czech Republic): Interaction of Receptor Tyrosine Kinases with the Primary Cilium
11. Pařízková Martina (Prague, Czech Republic): Early Diagnosis of Alzheimer´s Disease Using Spatial Navigation Assessment
12. Peltanová Barbora (Brno, Czech Republic): You Scratch My Back; I'll Ride on Yours: Metabolic Manipulation in HNSCC Stroma
13. Samuel Sabrina (Hull, United Kingdom): The Role of Arginine Methylation in Glioblastoma
SCIENTIFIC PROGRAMME
14. Schmidt Jan (Hradec Králové, Czech Republic): Low Molecular Weight Hyaluronic Acid Effect on Dental Pulp Stem Cells
15. Součková Olga (Prague, Czech Republic): Screening of Low-Molecular Inhibitors of Purine Synthesis and Their Use in Treatment of Purine Synthesis Disorders and Cancer
16. Svatoň Michal (Prague, Czech Republic): A Homozygous Deletion in the SLC19A1 Gene as a Cause of Folate-Dependent Recurrent Megaloblastic Anemia 17.
Szabo Petra Lujza (Vienna, Austria): Accompanied with Cardiac Fibrosis in a Mouse Model of Duchenne Muscular Dystrophy
18.
Toman Daniel (Ostrava, Czech Republic): Effect of Bariatric Surgery on Fatty Liver Disease: A Prospective One Year Follow-Up Study
19.
Vašendová Veronika (Ostrava, Czech Republic): The Influnce of Transcutaneous Vagus Nerve Stimulation on Memory - a Pilot Study
20.
Vočková Petra (Prague, Czech Republic): Anti-Angiogenic Therapy with Bevacizumab is Effective In Vivo in Ibrutinib-Resistant Mantle Cell Lymphoma 21.
22. Vozandychová Věra (Hradec Králové, Czech Republic): Changes of
DeubiquitinatingEnzymes upon Infection by Intracellullar Bacteria Francisella Tularensis
Szabó Dóra (Pécs, Hungary): Potential Cardioprotective Effect of Pituitary Adenylate Cyclase Activating Polypeptide in Heart Failure
EVALUATION COMMITTEE
Chairperson: Miroslav Červinka Dean Emeritus
Charles University, Faculty of Medicine in Hradec Králové Czech Republic
Members: Margarethe Geiger
Head of Department of Vascular Biology and Thrombosis Research Centre of Physiology and Pharmacology Medical University of Vienna, Austria
John Greenman
Professor of Tumour Immunology
Head of School of Biological, Biomedical and Environmental Sciences University of Hull, United Kingdom
Peter Soeters
Professor Emeritus of Surgery (Gastroenterology and Clinical Nutrition) Former President of the International Association for Surgical Metabolism and Nutrition (IASMEN)
Chairman of the European Society of Parenteral and Enteral Nutrition (ESPEN)
Maastricht University Medical Centre, the Netherlands Gabor Kovacs
Professor Emeritus
A Regular Member of the Hungarian Academy of Sciences
Chairman of the Doctoral Council of the Hungarian Academy of Sciences Faculty of Medicine, University of Pécs, Hung
Jan Škrha
Vice-Rector for International Affairs Charles University, Prague, Czech Republic
Study of the secretion of outer membrane vesicles in the bacterium F. tularensis and their role in the host - pathogen interaction
Jan Bavlovič
Department of Molecular Pathology and Biology, Faculty of Military Health Sciences, University of Defence, Czech Republic
Tutors: J. Klimentová
Introduction
The genus Francisella are encapsulated immobile gram-negative cocobacilli that are also intracellular pathogens of humans and many animals. This genus consists of three species: F. tularensis, F. novicida and F. philomiragia, but only the first one is clinically relevant. F. tularensis is highly infectious and potentially causes a febrile condition known as tularemia (Celli and Zahrt, 2013). The potential misuse of F. tularensis as a biological weapon has led disease control and prevention centers to classify the bacterium as a category A substance (Celli and Zahrt, 2013).
Although Francisela exhibits an extracellular phase during infection, its survival and replication inside host cells is considered a key aspect of its life cycle. This is evidenced by its ability to enter, survive and proliferate in various types of host cells, including macrophages, dendritic and polymorphonuclear cells (Celli and Zahrt, 2013).
Bacterial OMVs are spherical particles with a size of 20-200 nm, which are formed by bulging and separation of part of the outer membrane. OMVs are produced by all gram-negative bacteria and are considered to be one of the major secretory pathways. Bacteria use them in the interaction with both other bacterial cells and eukaryotic (host) cells. In recent studies (McCaig et al., 2013, Klimentova et al., 2019) it has been shown that Francisella produces, in addition to classical spherical OMVs, also membrane vesicles of unusual tubular shape. The aim of the presented study was to contribute to our current knowledge about the role of Francisella OMVs in the host-pathogen interaction. We also studied the secretion of OMVs in several mutant Francisella strains with disrupted surface (on the level of lipopolysaccharide O-antigen) with lower abilities to form the tubular OMVs.
Methods
We studied the levels of a wide range of proinflammatory cytokines (IL-1, IL-6, IL12-p70, TNFα, VEGF-A, CXCL- 1, MCP1, GM-CSF) and anti-inflammatory cytokine (IL-10) after administration of OMVs, bacteria and their combinations to murine bone marrow derived macrophages (BMDM). OMVs were added to BMDM at different doses, and at different time intervals the supernatant was collected, in which the levels of the cytokines and chemokines corresponding to the inflammatory response were determined by ELISA.
The phenotype of the mutant strains with disrupted O-antigen was monitored by the growth curves, the vesiculation rates and scanning electron microscopy (SEM), and the isolated OMVs were also visualized by transmission electron microscopy (TEM). Their sensitivity to the complement contained in the serum was evaluated by the serum killing assay.
Results
OMVs induced massive production of proinflammatory cytokines in BMDM, while the bacterium alone elicited no proinflammatory response. On the other hand, the strong proinflammatory activation of macrophages by OMVs was neither suppressed by their pretreatment by the bacterium, nor was it reverted by the bacterium added post-OMVs.
Some of the mutant strains with disrupted lipopolysaccharide (LPS) showed elevated vesiculation rates compared to the wild type strain. According to TEM and SEM we observed also differences between mutant strains in the shapes of vesicles as well as in the formation of nanotubular protrusions and connection threads between adjacent bacteria.
The parent virulent wild type strain of F. tularensis showed also complete resistance to serum complement, while the LPS-disrupted mutant strains were highly sensitive to this complement.
Discussion
OMVs in Francisella are known to contain enormous number of previously reported virulence factors and immunomodulatory proteins. As such they are expected to play some role in the host-pathogen interaction and they are also interesting from the point of view of the potential subunit vaccine development. Our results suggest that while the bacterium has some effective mechanisms to suppress the natural response of the host cell leading to inflammation and killing of the bacterium, the isolated OMVs show exactly the opposite reaction. The proinflammatory response to OMVs could not be reverted by the bacterium. The exact mechanism by which OMVs interfere with the inflammation machinery is not clear.
In the second part of the study we focused on the unusual tubular shape of Francisella OMVs. OMVs originate from the bacterial surface so we selected several mutant strains with disrupted surface and studied their vesiculation. From a larger number of various mutants we further picked a small group of mutants lacking various genes involved in the biosynthesis of O-antigen and LPS. Some of these strains exhibited hypervesiculation, nevertheless, they were unable to form tubular vesicles and tubular protrusions or this ability was diminished. O-antigen is the building block not only of LPS but also of the polysaccharide capsule in Francisella. We observed some correlation between the different ability of the mutant strains to form the capsule and their ability to form nanotubes. Similarly, their susceptibility to killing by the serum complement was much higher than in the wild type strain. LPS of F. tularensis is unique compared to other bacteria not only regarding its structure but mainly by its inability to elicit proinflammatory response (Gunn and Ernst, 2007). We here propose that either the unique structure of Francisella LPS or the polysaccharide capsule relates somehow with the unique shape of its OMVs.
Conclusions
Isolated OMVs from F. tularensis elicit strong proinflammatory response in bone marrow derived macrophages while the parent bacterium itself suppresses it. The mechanism by which OMVs interfere with the inflammation cascade is not clear and more research has to be done to reveal their exact role in the interaction with the host.
Regarding the unique tubular shape of Francisella OMVs, we believe, that it is related to the unique structure of its LPS and polysaccharide capsule.
Summary
The aim of this work was to describe the interaction of outer membrane vesicles (OMVs) isolated from F. tularensis with a model host cell (bone marrow macrophages). Changes in cytokine levels and direct action on host cells were
evaluated. We also studied mutant strains of F. tularensis with impaired formation of surface structures, especially lipopolysaccharide, in which a different vesicle morphology and sensitivity to complement was confirmed.
References
Klimentova, J., Pavkova, I., Horcickova, L., Bavlovic, J., Kofronova, O., Benada, O., & Stulik, J. (2019). Francisella tularensis subsp. holarctica Releases Differentially Loaded Outer Membrane Vesicles Under Various Stress Conditions. Frontiers in Microbiology, 10. doi:10.3389/fmicb.2019.02304
Celli, J., and Zahrt, T.C. (2013). Mechanisms of Francisella tularensis intracellular pathogenesis. Cold Spring Harb.
Perspect. Med. 3, a010314
McCaig, W.D., Koller, A., and Thanassi, D.G. (2013). Production of outer membrane vesicles and outer membrane tubes by Francisella novicida. J. Bacteriol. 195, 1120–1132.
Gunn, J. S., & Ernst, R. K. (2007). The Structure and Function of Francisella Lipopolysaccharide. Annals of the New York Academy of Sciences, 1105(1), 202–218. doi:10.1196/annals.1409.006
Additional information
Year of Graduation (Master’s degree): 2016 Year of beginning Ph.D. studies: 2017
Topic of Ph.D. dissertation: Study of the secretion of outer membrane vesicles in the pathogen F. tularensis and their role in the host - pathogen interaction
Role of the author and co-authors in preparing and carrying out the research included in the presentation: J.B.
prepared and carried out the experiments, evaluated and interpreted the data and wrote the manuscript; J.K. designed the study, interpreted the data and discussed the results.
The impact of proton pump inhibitors on direct oral anticoagulants activity in patients with atrial fibrillation
Tomáš Bolek
(1) Department of of Internal Medicine I, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovak Republic.
(2) Department of Hematology and Blood Transfusion, National Centre of Hemostasis and Thrombosis, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovak Republic
Co-authors: M. Samoš (1), P. Galajda(1), J.Staško (2), M. Mokáň (1) Tutor: M. Samoš
Introduction
Direct oral anticoagulants (DOACs) are frequently used for long-term pharmacological prevention of stroke or systemic embolism in patients with atrial fibrillation (1). However, such long-term DOAC therapy significantly increases the risk of upper gastrointestinal (GI) bleeding. Proton pump inhibitors (PPIs) administrated together with DOACs therapy may reduce the risk of GI bleeds. Nevertheless, recently published studies discuss possible
interaction between PPIs and DOAC therapy (2).
The aim of our study was to determine the impact of PPIs on DOACs activity in patients with non-valvular atrial fibrillation (NV-AF).
Methods
A preliminary, prospective, observational, clinical study in patients with NV-AF on long-term DOACs (dabigatran, rivaroxaban, apixaban) therapy was performed. 139 presentations of patients (64 men, 75 women, mean age 75 years, ranging from 35 to 92) with NV-AF treated with DOACs were enrolled to this study.
Exclusion criteria for patient enrollment included a recent or pending surgery, recent or known bleeding disorders, disabling or recent stroke, uncontrolled hypertension, need for anticoagulation for disorders other than NV-AF, planned ablation or surgery for NV-AF, severe renal dysfunction (creatinine clearance ≤ 30 mL/min), active liver disease and extreme body weight (body mass index ≤ 18 and ≥ 40 kg/m2). DOACs were taken in two dose regimen dabigatran (110 mg/twice daily: 34 patients, 150 mg/twice daily: 27 patients), rivaroxaban (20 mg/once daily: 12 patients, 15 mg/once daily: 30 patients), apixaban (5 mg/twice daily: 17 patients, 2.5 mg/twice daily: 19 patients). PPIs were co-administrated in 86 patients (dabigatran: 42 patients. rivaroxaban: 23 patients, apixaban: 21 patients). In dabigatran-treated patients, 22 patients were treated with omeprazole (20 mg /twice daily) and 20 patients with pantoprazole (40 mg/once daily) and PPI treatment was withdrawn for a period of 2 weeks in 23 patients on long term dabigatran/PPI co-therapy, and dabigatran levels were re-tested after PPI withdrawal (a washout study was performed). Samples were taken for assessment of trough and peak levels on fifth day of in-hospital stay. Dabigatran levels (ng/mL) were assessed with Hemoclot Thrombin Inhibitor Assay and rivaroxaban and apixaban levels (ng/mL) with factor Xa-calibrated anti-Xa chromogenic analysis.
Results
There were no significant differences in basic demographic data and concomitant medication in patients with and without PPI therapy. The analysis of dabigatran levels showed that there were significant differences in dabigatran trough levels comparing patients treated with PPI and patients without PPI (58.86 ± 36.76 ng/mL in PPI group vs. 110.72 ± 88.47 ng/mL in non-PPI group, p < 0.05) Similarly, there were significant differences in dabigatran peak levels between compared groups (88.0 ± 20.5 ng/mL in PPI group vs. 174.4 ± 139.64 ng/mL in non-PPI group, p < 0.05). Moreover, when omeprazole-treated and pantoprazole-treated patients were compared, no significant differences in dabigatran trough (62.4 ± 51.1 ng/mL versus 60.3 ± 53.1 ng/mL; p = 0.92) or peak (116.4 ± 51.2 ng/mL versus 110.1 ± 49.2 ng/mL; p = 0.79) levels were found. Subsequently, the analysis of dabigatran levels after 2 weeks of PPI withdrawal demonstrated a significant increase in trough levels (97.2 ± 79.7 ng/mL when PPI was taken vs. 163.8 ± 105.5 ng/mL when PPI was withdrawn; p < 0.05). Similar increase was seen in dabigatran 110 mg twice-daily treated patients (110.4 ± 80.3 ng/mL on PPI vs. 155.1 ± 107.9 ng/mL without PPI/; p < 0.05) and dabigatran 150 mg twice-daily-treated patients (89.7 ± 50.1 ng/mL on PPI vs. 168.8
± 108.0 ng/mL without PPI; p < 0.001). Furthermore, the analysis of anti-Xa levels did not show significant
differences in trough rivaroxaban anti-Xa levels (53.6 ± 41.5 in PPI vs. 34.9 ± 19.3 ng/mL in non-PPI group, p = 0.09), and trough apixaban anti Xa activity (102.4 ± 75.4 in PPI vs. 122.4 ± 70.1 ng/mL in non-PPI group, p = 0.43) according the PPI status. Similarly, there were no significant differences in peak rivaroxaban anti-Xa levels (189.9 ± 116.8 vs. 186.3 ± 97.2 ng/mL, p = 0.43) and peak apixaban anti-Xa levels (162.5 ± 39.1 vs. 223.2 ± 62.4 ng/mL, p = 0,09) comparing PPI and non-PPI treated patients.
Discussion
DOACs significantly increase the risk of GI bleeding, but it was demonstrated that PPIs may reduce the risk of this bleeding (2). Therefore, DOACs and PPIs are relatively often co-administrated, mainly in patients with high risk of GI bleeding. However, there is a discussion about possible interaction between PPIs and DOAC therapy that may reduce the DOAC activity (2). The most probable interaction is between dabigatran and PPI, due to pharmacological properties of dabigatran, such as a pH dependent solubility, the fact that dabigatran etexilate is a substrate for P-glycoprotein efflux transporter (P-gly) and PPIs increase gastric pH, are substrates and inhibitors of P-gly (3). Our study demonstrated significant differences in dabigatran trough and peak levels according to PPI status (4), confirming suggested dabigatran/PPI interaction. This priority observation was confirmed by Japanese authors, who, in a randomized study, showed that PPIs significantly reduced dabigatran trough and peak levels (5). The most probable mechanism of this interaction is an interaction through the effect on gastric pH (as the absorption of dabigatran etexilate is pH dependent). However, our study does not give direct confirmation of this mechanism, as we did not measure plasma gastrin levels or the pH of gastric juice in our study. Therefore, this mechanism is supported only indirectly by the pharmacologic profile of dabigatran etexilate and by our previous observation of low dabigatran trough and peak levels in patient after total gastric removal (with no gastric acid secretion) despite full dabigatran etexilate dosing (6). Additionally, it was not entirely clear whether the interaction between PPI and dabigatran etexilate is a ”class effect´´ or a ”drug effect´´.
In our study we did not show differences in dabigatran trough and peak levels between omeprazole-treated and pantoprazole-treated patients with NV-AF (7). This observation corresponds with one reported in previously mentioned study on dabigatran-treated Japanese patients, in whom similar effect on dabigatran levels was observed with esomeprazole, lansoprazole, and rabeprazole (5). Furthermore, this study showed a significant increase in dabigatran trough levels after 2 weeks of PPI withdrawal (8). This observation probably confirms that a PPI/dabigatran interaction exists, and points on the reversibility of this interaction, which may be important for clinical practice. Finally, we did not demonstrate significant differences in anti-Xa xabans (rivaroxaban, apixaban) levels in patients with NV-AF according to PPI status (9).
Conclusion
This pilot study did not reveal significant changes in xabans anti-Xa levels according the PPI status, but reveal significant changes in dabigatran anti-IIa levels according to PPI status.
Summary:
Proton pump inhibitors (PPIs) administrated together with direct oral anticoagulants (DOACs) therapy may reduce the risk of gastrointestinal (GI) bleeding. Recently published studies discuss a possible interaction between PPIs and DOACs therapy. The aim of this study was to determine the impact of PPIs on DOACs plasma levels in patients with non-valvular atrial fibrillation (NV-AF). We performed a prospective, observational study in patients with NV-AF on long term DOACs therapy. We enrolled 139 patients (64 men, 75 women, mean age 75 years) with NV-AF treated with DOACs (61 patients: dabigatran, 42 patients: rivaroxaban, 36 patients:
apixaban). PPIs were co-administrated in 86 patients (dabigatran: 42 patients. rivaroxaban: 23 patients, apixaban:
21 patients). Samples were taken for assessment of trough and peak DOACs levels. Dabigatran levels (ng/mL) were assessed with Hemoclot Thrombin Inhibitor Assay and rivaroxaban and apixaban levels with factor Xa- calibrated (ng/mL) anti-Xa chromogenic analysis. In dabigatran-treated patients, PPI were withdrawn for a period of 2 weeks and than dabigatran levels were re-assessed. The analysis of dabigatran levels showed that there were significant differences in dabigatran trough levels comparing patients treated with PPI and patients without PPI (58.86±36.76 in PPI group vs. 110.72± 88.47 ng/mL in non-PPI group, p<0.05) Similarly, there were significant differences in dabigatran peak levels between compared groups (88.0±20.5 in PPI group vs.
174.4±139.64 ng/mL in non-PPI group, p<0.05). Subsequently, the analysis of dabigatran plasma levels after 2 weeks of PPI withdrawal demonstrated significant increase in trough dabigatran levels. Furthermore, the analysis of anti-Xa xabans levels did not show significant differences in trough and peak anti-Xa rivaroxaban and apixaban levels according to PPI status. Concluding, our study revealed significant differences in dabigatran levels according to PPI status, but no such differences were found in xabans-treated patients.
Key words: proton pump inhibitors, direct oral anticoagulants, atrial fibrillation
References
1. Bolek T., Samoš M., Staško J., Mokáň M.. Antitrombotická liečba v kardiológii akútny koronárny syndróm, fibrilácia predsiení, venózna tromboembolická choroba a diabetes mellitus : vysokoškolsá učebnica; P+M 2020 ISBN 978-80-89694-73-0
2. Bolek T, Samoš M, Škorňová I, Kovář F, Galajda P, Staško J, Kubisz P, Mokáň M. Proton Pump Inhibition in Patients Treated With Novel Antithrombotic Drugs: Should We Worry About Thrombosis?; J Cardiovasc Pharmacol. 2018 Jul;72(1):71-76.
3. Bolek T, Samoš M, Škorňová I, Galajda P, Staško J, Kubisz P, Mokáň M.Proton Pump Inhibitors and Dabigatran Therapy: Impact on Gastric Bleeding and Dabigatran Plasma Levels; Semin Thromb Hemost . 2019 Nov;45(8):846-850.
4. Bolek T, Samoš M, Stančiaková L, Ivanková J, Škorňová I, Staško J, Galajda P, Kubisz P, Mokáň M.
The Impact of Proton Pump Inhibition on Dabigatran Levels in Patients With Atrial Fibrillation;Am J Ther. 2019 May/Jun;26(3):e308-e313.
5. Kuwayama T, Osanai H, Ajioka M, Tokuda K, Ohashi H, Tobe A, Yoshida T, Masutomi T, Kambara T, Inoue Y, Nakashima Y, Asano H, Sakai K. Influence of proton pump inhibitors on blood dabigatran concentrations in Japanese patients with non-valvular atrial fibrillation. J Arrhythm 2017; 33: 619 - 623.
6. Bolek T, Samoš M, Škorňová I, Stančiaková L, Korpallová B, Galajda P, Staško J, Kubisz P, Mokán M.
How to proceed with long-term anticoagulation in patient after total gastrectomy and atrial fibrillation?;
Eur J Clin Pharmacol. 2019 Feb;75(2):285-286.
7. Bolek T, Samoš M, Škorňová I, Schnierer M, Lipták P, Bánovčin P, Urban L, Staško J, Kubisz P, Galajda P, Mokán M.Dabigatran levels in omeprazole versus pantoprazole-treated patients with atrial fibrillation: is there a difference?; Eur J Clin Pharmacol. 2019 Jun;75(6):875-877.
8. Schnierer M, Samoš M, Bolek T, Škorňová I, Nosáková L, Bánovčin P, Galajda P, Stasko J, Kubisz P, Hyrdel R, Mokáň M.The Effect of Proton Pump Inhibitor Withdrawal on Dabigatran Etexilate Plasma Levels in Patients With Atrial Fibrillation: A Washout Study; J Cardiovasc Pharmacol. 2020 Apr;75(4):333-335.
9. Bolek T, Samoš M, Škorňová I, Stančiaková L, Staško J, Korpallová B, Galajda P, Kubisz P, Mokáň M.Does proton pump inhibition change the on-treatment anti-Xa activity in xabans-treated patients with atrial fibrillation? A pilot study; J Thromb Thrombolysis. 2019 Jan;47(1):140-145.
Additional important information about the author:
Year of Graduation: 2015
Year of beginning Ph.D. studies: 2016
Topic of Ph.D. dissertation: The impact of proton pump inhibition on efficacy of antithrombotic therapy in patients with cardiovascular diseases.
Role of the author and co-authors in preparing and carrying out the research included in the presentation:
Tomáš Bolek: postgraduate student in Internal medicine. He designed and performed the study and drafted all parts of the presentation.
Matej Samoš: Associate - professor of Internal Medicine. He was responsible for the complex study as a tutor and contributed to the preparation of the presentation, supervision and the final approval.
Peter Galajda: Professor of Internal Medicine. He revised the abstract and presentation for important intellectual content and gave the final approval.
Ján Staško: Professor of Internal Medicine – Hematology, Head of the Dpt. of Hematology and Blood Transfusion. He was responsible for DOACs levels assessment, co-designed the study, revised the abstract and presentation for important intellectual content and gave the final approval.
Marián Mokáň: Professor of Internal Medicine, Head of the Dpt. of Internal Medicine I. He supervised the research, revised the abstract and presentation for important intellectual content and gave the final approval.
ALTERED IMMUNE RESPONSE AND IMPLANTATION FAILURE IN PROGESTERONE-INDUCED BLOCKING FACTOR-DEFICIENT MICE
Tímea Csabai
University of Pécs, Medical School, Department of Medical Biology and Central Electron Microscope Laboratory, Pécs, Hungary
Tutor: Júlia Szekeres-Barthó
Introduction
The success of embryo implantation depends on both embryo quality and the receptivity of the maternal endometrium. The progesterone-induced blocking factor (PIBF) is a downstream mediator, which conveys some of the immunological effects of progesterone. PIBF is required for the establishment and maintenance of pregnancy, both in humans and mice. In the sera of pregnant women, PIBF concentrations increase throughout gestation and drop before labour. During spontaneous miscarriage or preterm delivery, serum PIBF concentrations fall below the normal levels (1). PIBF deficiency results in increased resorption rates in pregnant mice, together with an inversion of the Th1/Th2 cytokine balance (2). Recent data show that PIBF plays a role in implantation in mice. PIBF induced the decidual transformation of mouse endometrial stromal cells, and PIBF expression peaks in the mouse endometrium during the implantation window (3). The immunological effects of PIBF play a role in establishing a favourable immunological milieu for the developing foetus. PIBF induces an increased synthesis of Th2 cytokines (4). Despite the presence of cytotoxic enzymes their cytoplasmic granules, the decidual NK cells are weakly cytotoxic. Earlier we showed that PIBF inhibits the degranulation of NK cells (5). A high number of mouse decidual NK cells that contain PIBF in their cytoplasmic granules, suggest that the local presence of PIBF might be a factor in the low decidual NK activity (6). In the present study, we aimed to investigate the consequences of anti-PIBF treatment of pregnant mice during the peri- implantation period on reproductive performance as well as the underlying mechanisms.
Experimental design
CD1 female mice were injected i.p., with 2μg anti-PIBF monoclonal antibody on days 1.5 and 4.5 of pregnancy.
The number of implantation sites and resorption rates were recorded on day 10.5. PIBF+ decidual NK cells and B cells were detected with immunohistochemistry or immunofluorescence. Decidual and peripheral NK activity was assessed by flow cytometry. A prime PCR array was used for determining differential expression of genes involved in lymphocyte activation and Th1 or Th2 differentiation, in CD4+ and CD8+ spleen cells from pregnant anti-PIBF treated and control mice.
Results
While the average number of implanted embryos was 6.5 in the controls, in females treated with anti- PIBF antibody in the peri- implantation period, the mean implantation sites decreased to four. The 2% resorption rate in the control group increased to 40% in the functionally PIBF-deficient mice.
We found a significantly decreased number of PIBF+ NK cells in the day 10.5 deciduae of resorbed embryos from anti-PIBF-treated mice compared to normal deciduae from untreated mice.
Both the decidual and the peripheral NK activity of the anti-PIBF-treated mice were significantly (P < 0.05) increased compared to those of the controls.
Figure 1A Anti-PIBF treatment of pregnant mice in the peri-implantation period results in decreased implantation and increased resorption rates. CD1 mice were injected with anti-PIBF monoclonal antibody at days 1.5 and 4.5 of pregnancy. The controls were treated with PBS, among the same conditions. The number of implantation sites (1st panel) and resorption rates (2nd panel) were recorded on day 10.5. The implantation sites were significantly lower, while the resorption rates significantly increased in the anti-PIBF-treated mice. The columns represent the mean ±} SEM of the results from 10 (anti-PIBF-treated) and 15 (control) mice. *P < 0.05. B Immunohistochemical analysis of PIBF+ NK cells in the deciduae of normal (a) and anti-PIBF-treated (b) mice on day 10.5 of pregnancy (×400). C Cytotoxic activity of decidual and peripheral lymphocytes from anti-PIBF-treated and control mice on day 10.5 of pregnancy. The cytotoxic activity of peripheral and decidual NK cells from control and anti-PIBF-treated mice was determined by flow cytometric analysis of target cell damage on a single-cell level. The target cells were labelled with PKH-67 and stained with propidium iodide after 4 h of incubation with the lymphocytes to distinguish apoptotic from non-apoptotic target cells. The bars represent the mean ±} SEM of at least six experiments. *P <
0.05.
The endometria of the control animals contained decidual NK cells and a discrete layer of B cells at the choriodecidual interface. While the NK cells were still present, the B cells disappeared from the deciduae of the resorbed embryos from the anti-PIBF treated mice.
Figure 2 Decidual B cells in control (A) and anti-PIBF-treated (B) mice. B cells were reacted with rat anti-mouse B220 IgG conjugated with Alexa Fluor 647 (red fluorescence), and NK cells were reacted with fluorescein-conjugated DBA lectin (green fluorescence). (A) Decidua of an untreated mouse. NK cells (a, c) are present in the decidua and B cells (b, c) are located at the choriodecidual interface. (B) Decidua of anti-PIBF treated mouse. NK cells (a, c) are present, while B cells (b, c) are absent. (a) NK cells, (b) B cells, and (c) merged (×200).
The genes implicated in T cell activation, e.g., members of the CD3 complex (CD 247, CD3D, CD3E, CD3G, and IL2RG), were significantly downregulated in CD4+ spleen cells of anti-PIBF-treated mice but significantly increased in CD8+ cells of the same animals. In the anti-PIBF-treated mice, the beta chain of IL2R was downregulated in the CD4+ population, while in the CD8+ population the alpha and the gamma chain of IL2R and IL2 increased. The genes of the co-stimulatory molecules were altered in a similar fashion. Upon anti-PIBF treatment, the genes for CD4, CD28, CD40L, and CD86 were downregulated in the CD4 and upregulated in the CD8 population. These data suggest that the absence of PIBF inhibits the activation of CD4+ cells and facilitates that of CD8+ T cells. Among genes involved in Th1/Th2 pathway, IL-4 was significantly downregulated in CD4+
cells of the anti-PIBF treated mice. At the same time, IL-12 was upregulated in CD8+ cells of the anti-PIBF-treated animals.
Figure 3 A Differential expression of genes implicated in T cell activation in splenic CD4+ and CD8+ T spleen cells of anti-PIBF-treated mice and controls. Heatmap of the T cell activation-related mRNA expression of genes in CD4+ and CD8+ splenocytes of anti-PIBF-treated and control mice. Clear separations are seen between the anti-PIBF-treated and control animals and also between the CD4+ and CD8+ cell types. Members of the CD3 complex and co-stimulatory molecules were downregulated in CD4+ cells and upregulated in CD8+ cells of anti-PIBF-treated mice. All of the results shown were significantly (P < 0.05) different from the values of the controls. The expression intensities were scaled on rows (genes) to Z scores to make them weigh equally in the clustering. The colours of the heatmap are mapped linearly to the Z scores (low expression in green and high expression in red). B Differential expression of genes involved in Th1/Th2 differentiation by spleen cells of control and anti-PIBF-treated mice. (a) IL12A mRNA is significantly upregulated in CD8+ cells of anti- PIBF-treated mice. (b) IL-4 mRNA is significantly downregulated in CD4+ cells of anti-PIBF-treated mice. The bars represent the mean ±}
SEM of four experiments. *P < 0.05.
Discussion
PIBF induces decidual transformation of mouse endometrial stromal cells, and PIBF levels in the endometrium increase during the implantation window the (3), suggesting that PIBF might play an active role in implantation.
To confirm this hypothesis, we neutralized the biological activity of PIBF during the peri-implantation period in mice and investigated the consequences of functional PIBF deficiency at several levels. Anti-PIBF treatment of pregnant mice at days 1.5 and 4.5 of pregnancy resulted in a significantly reduced number of the implantation sites, and the implantations that took place, nevertheless, were compromised. Failed pregnancies are characterized by high peripheral NK activity, both in humans and in mice (6). Decidual NK cells, which constitute 60% of decidual lymphocytes have a low cytotoxic activity, despite the availability of perforin and granzyme in their cytoplasmic granules (7). PIBF blocks the upregulation of perforin expression in activated decidual lymphocytes and inhibits NK cell cytotoxicity by blocking granule exocytosis (5). Here we show that anti-PIBF treatment during the peri- implantation period results in the reduced presence of PIBF+ NK cells in the day 10.5 decidua, together with significantly increased decidual and peripheral NK activity, compared to the controls. Earlier studies showed that increased resorption rates in anti-PIBF-treated mice were corrected by simultaneous treatment of the mice with anti-NK antibodies (8), suggesting that PIBF prevents pregnancy loss in mice—at least partly—by blocking NK activity. Increased decidual NK activity owing to the loss of PIBF+ decidual NK cells could be one of the
reasons for the increased resorption rates in the anti-PIBF treated mice. B cells constitute a minor population among decidual lymphocytes, yet they might be important for the immunological balance of the decidua. A recent study showed that the IL-33- induced expression of PIBF1 by decidual B cells protects against preterm labour both in humans and in mice (9). In the present study, B cells were depleted from the deciduae of PIBF deficient mice.
Taken together, it is conceivable that anti-PIBF treatment—by depleting decidual B cells—will, at a later stage, put pregnancy at risk due to the lack of PIBF-positive B cells. Finally, we performed a gene array on spleen cells of anti- PIBF-treated and control mice in order to investigate whether the absence of functional PIBF influenced T cell activation and differentiation. In this study, we found that members of the T cell receptor CD3 complex were significantly downregulated on CD4+ T cells of anti-PIBF-treated mice, while CD3D and IL2R B and G were upregulated in CD8+ cells, suggesting that Th cell activation is severely inhibited in the anti-PIBF-treated pregnant mice.
IL-4 is the main cytokine driving Th2 cell differentiation (10). In our hands, the gene for IL-4 was significantly downregulated in CD4+ cells, while that of IL-12A was upregulated in CD8+ cells of the anti-PIBF-treated mice.
There is evidence that the recognition of paternal antigens by the maternal immune system is not simply harmless but necessary for the setting in of the mechanisms that adapt the immune response to tolerate the foetus. Following recognition of foetal antigens, the immune system becomes activated, and this results in the establishment of regulatory mechanisms, e.g., a Th2 dominant immune response (10). Neutralizing PIBF in the peri-implantation period abolishes this mechanism right at the start. CD4+ T cell activation is disturbed, T cells differentiate in the Th1 direction, and as a result, implantation as well as ongoing pregnancies is compromised.
Conclusion/ Summary
These data suggest that the lack of PIBF results in an impaired T cell activation, together with a Th1 differentiation, and increased NK activity, resulting in implantation failure.
Funding:
This work was supported by GINOP-2.3.2-15-201600021, ÁOK KA Research Grants (KA 2017-22 and KA-2018- 07), EFOP- 3.6.1.-16-2016-00004, and OTKA K125212.
References
1. Polgár B, Nagy E, Mikó E, Varga P, Szekeres-Barthó J. Urinary progesterone induced blocking factor concentration is related to pregnancy outcome. Biol Reprod. (2004) 71:1699–705.
2. Szekeres-Bartho J, Par G, Szereday L, Smart CY, Achatz I. Progesterone and non-specific immunologic mechanisms in pregnancy. Am J Reprod Immunol. (1997) 38:176–82.
3. Mulac-Jeriˇcevi´c B, Šu´curovi´c S, Gulic T, Szekeres-Bartho J. The involvement of the progesterone receptor in PIBF and Gal-1 expression in the mouse endometrium. Am J Reprod Immunol. (2019) 81:e13104.
4. Szekeres-Bartho J, Wegmann TG. A progesterone-dependent immunomodulatory protein alters the Th1/Th2 balance. J Reprod Immunol. (1996) 31:81–95.
5. Faust Z, Laskarin G, Rukavina D, Szekeres-Bartho J. Progesterone-induced blocking factor inhibits degranulation of natural killer cells. Am J Reprod Immunol. (1999) 42:71–5.
6. Bogdan A, Berta G, Szekeres-Bartho J. PIBF positive uterine NK cells in the mouse decidua. J Reprod Immunol. (2017) 119:38–43.
7. Rukavina D, Rubesa G, Gudelj L, Haller H, Podack ER. Characteristics of perforin expressing lymphocytes within the first trimester of human pregnancy. Am J Reprod Immunol. (1995) 33:394–404
8. Szekeres-Bartho J, Par G, Dombay Gy, Smart YC, Volgyi Z. The antiabortive effect of progesterone- induced blocking factor in mice is manifested by modulating NK activity. Cell Immunol. (1997) 177:194–
9.
9. Huang B, Faucette AN, Pawlitz MD, Pei B, Goyert JW, Zhou JZ, et al. Interleukin-33-induced expression of PIBF1 by decidual B cells protects against preterm labor. Nat Med. (2017) 23:128–35.
10. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol Today. (1993) 14:353–6.
Additional information:
Year of Graduation (Master’s degree): 2013 Year of beginning Ph.D. studies: 2013
Topic of Ph.D. dissertation: Examination of factors affecting the implantation.
Role of the author and co-authors in preparing and carrying out the research included in the presentation: TC:
performed the work. JS-B designed and supervised the experiment.
DIFFERENTIALLY EXPRESSED MITOCHONDRIAL PROTEINS IN MULTI-STEP SELECTED TAXANE-RESISTANT MCF-7 BREAST
CANCER CELLS
Petr Daniel
Department of Biochemistry, Cell and Molecular Biology, Charles University Third Faculty of Medicine in Prague, Czech Republic
Co-authors: P. Halada, M. Jelínek
Tutor: J. Kovář
Introduction
The lack of response of tumour cells to initially efficient therapy with any antineoplastic drug, the acquired resistance, is a major cause of high cancer mortality. Thus, understanding the mechanism(s) of acquired drug resistance is a major concern.
Previously, we reported ABC-transporters expression analysis of paclitaxel-sensitive MCF-7 and paclitaxel-resistant MCF-7/PacR breast cancer cells (1). ABCB1 plasma membrane transporter was found to be overexpressed in MCF- 7/PacR cells (1), in MCF-7/SB-T-0035R cells (2), and recently, in ovarial cancer cells resistant to paclitaxel (our unpublished data). Further, we have shown changed expression of CTSD, TRIP6 and HSPB1 genes in MCF-7/PacR cells using the two-dimensional electrophoresis (2D-PAGE) approach (3).
Mitochondria play a crucial role in cell metabolism, and particularly, in regulated cell death to taxanes. Thus, adaptation of tumour cells to taxanes may involve mitochondrial changes as well. Thus, the aim of the project was to find and determine differentially expressed mitochondrial proteins between MCF-7 and MCF-7/PacR cells. To achieve this goal, we carried out the 2D-PAGE with mitochondria enriched fraction in combination with mass spectrometry analysis (MALDI-TOF, in colaboration with Dr. Halada). Next aim was to verify the expression of two suspected genes by immunobloting not only in MCF-7/PacR cells, but also in MCF-7/PacR100 and MCF-7/SB-T-0035R cells.
Moreover, we examined cell localization of carbamoyl-phosphate synthetase 1 (CPS1) in MCF-7 and MCF-7/PacR cells using the confocal microscopy.
Methods
All cell lines were cultivated in RPMI medium supplemented with 10 % FBS and streptomycin-penicilin mix.
MCF-7/PacR and MCF-7/SB-T-0035R cells have been established by step-wise mass population method of selection of parental MCF-7 cells in RPMI medium containing paclitaxel (Merck) (4), or Stony-Brooke Taxane (SB-T-0035, kindly provided by prof I. Ojima) (both 300 nM). MCF-7/PacR100 cells represent a previous step in the procces of selection of MCF-7/PacR cells (i.e., cultivated in 100 nM paclitaxel).
QProteome Mitochondria Isolation Kit (QIAGEN) was used for mitochondria enrichment following the manufacturer´s instructions. For 2D-PAGE, a pellet enriched for mitochondria was lysed in mild 2D-PAGE compatible PEB V buffer (GE Healthcare). Protein concentration was determined by 2-D Quant Kit (GE Healthcare). 150 µg of proteins were rehydrated for 24 hours, and separated at 65KV·h total voltage on 11-cm IPG strips (3-11NL) for 22 hours. The second dimension was performed in 12% hand-made polyacrylamide gel. Gels were stained in colloidal coomassie brilliant blue G250 overnight. Destained gels were scanned and spot analysis was perfomed using an ImageMaster® 2D Platinum software (GE Healthcare). Spots with two-fold change in spot volume was taken for MALDI analysis (kindly performed by Dr. Halada, Institute of Microbiology, Prague) (4).
Immunobloting was performed with proteins solubilized in RIPA buffer. Protein concentration was determined by BCA kit (Pierce). 20 µg of proteins were separated in 12% gel, blotted onto nitrocelullose membrane. Membrane was blocked with 5 % BSA and incubated with specific primary antibodies (anti-ABHD11, ab103130, 1:1000; anti-CPS1, ab129076, Abcam; 1:1000, anti-actin, clone AC-40, 1:1000, Merck) overnight. Secondary goat HRP-linked anti- mouse (sc-2005, 1:5000, Santa Cruz) and goat anti-rabbit (sc-2004, 1:5000) antibodies were used. The pictures were taken using a CCD camera GEL Logic 4000 (Carestream Health).
For confocal microscopy, seeded cells were fixed in 4% paraformaldehyde, washed, and permeabilized with 0.3%
Triton X-100. After the blocking step with 1% BSA, the cells were incubated with the primary antibodies diluted 1:200 in 1% BSA anti-CPS1 (ab129076) and anti-COX IV (#11967S, Cell Signaling Technology) overnight. Following secondary antibodies were used: goat anti-rabbit IgG H&L Alexa Fluor®488 (ab150077, Abcam) and goat anti-mouse IgG H&L F(ab)2 Alexa Fluor®594 (#8890S, Cell Signaling Technology). Next, cells were transferred onto a droplet of Vectashield® Mounting Medium with DAPI (Vector Laboratories) and sealed. Samples were analysed using a Leica TCS SP5 confocal microscope (Leica).
Results
Spots with changed spot volume are shown in Fig. 1. These spots were determined as the mitochondrial protein carbamoyl-phosphate synthetase 1 (CPS1, spot 5), abhydrolase domain-containing protein 11 (ABHD11, spot 3), adenylate kinase 2 (AK2, spot 7), ATPase family AAA domain-containing proteins 3A/3B (ATAD3A/3B, spot 6), and the light chain of cathepsin D (spots 1, and 2).
Fig. 1: Representative 2D-PAGE gels of mitochondria-enriched fraction of MCF-7 and MCF-7/PacR cells. Spots with a statistically significant change in spot volume are shown by arrows.
In particular, CPS1 (494 %) and ABHD11 (68 %) have significantly changed expression also in a control immunobloting analysis (Figure 2). Notably, expression of the ABHD11 gene was similarly changed in MCF- 7/PacR100 (146 %), and in MCF-7/SB-T-0035R cells (156 %) (Figure 2). Further, expression of the CPS1 gene was not changed in MCF-7/SB-T-0035R cells.
Fig. 2: Expression of ABHD11, CPS1 and β-actin (control) in whole-cell lysates of MCF-7/PacR100, MCF-7/PacR and MCF-7/SB-T-0035R cells.
CPS1 colocalized with a mitochondrial marker CoxIV in both MCF-7 and MCF-7/PacR cells (Figure 3). We distinguished the cells differing in CPS1 signal intensity, as ilustrated in Figure 3, as the cells with a green (extreme CPS1 expression), orange (medium CPS1 expression) and red (not expressing CPS1) signal. However, no clear cell population has been observed in FACS analysis (data not shown) (4).
Fig. 3: Colocalization of CPS1 (green) with CoxIV (mitochondrial marker, red) in DAPI-stained MCF-7 and MCF- 7/PacR cells.
Discussion
Cell fractionation is a powerfull tool in finding the proteins, whose expression are masked in whole-cell 2D-PAGE analysis. Nuclear genes encoding proteins localized in mitochondria, in particular CPS1 and ABHD11, were shown to have significant differential expression between MCF-7 and MCF-7/PacR cells.
Considering CPS1, an urea cycle enzyme, its abundant expression in a few parental MCF-7 cells, and in many MCF- 7/PacR cells, indicates for complex inner cell heterogeneity. How this inner heterogeneity contributes to drug resistance is not known. Other interesting question is how the CPS1 gene expression is regulated in MCF-7 cells.
The ABHD11 gene encodes for a mitochondrial matrix serine hydrolase (5) which expression is similarly increased in MCF-7/PacR100, and in MCF-7/SB-T-0035R cells, however, decreased in MCF-7/PacR cells. Yet, ABHD11 is differentially expressed in paclitaxel-resistant ovarial carcinoma cell line SKOV-3/PacR. The same expression pattern displayed the neighbouring HSPB1 gene (data not shown). Remarkably, the HSPB1 and ABHD11 genes are localized on q arm of chromosome 7, approximately 15 Mbp apart of the ABCB1 gene, in non-tumour cell karyotype. Thus, we suppose that observed change in gene expression of ABHD11 likely reflects the complex chromosomal events involved in the selection process.
Conclusion
We have found several genes encoding for mitochondrial proteins by 2D-PAGE approach. CPS1 was found to be overexpresed in MCF-7/PacR cells. However, we have shown that the high number of CPS1-positive MCF-7/PacR cells caused this difference. ABHD11 was the most downregulated gene in MCF-7/PacR cells. However, dramatical changes in ABHD11 expression in different cell models likely indicate on chromosomal events involved in the selection process for high ABCB1 expression.
Summary
Mitochondrial proteins, encoded by nuclear genes (CPS1, ABHD11), have changed expression in paclitaxel-resistant MCF-7/PacR breast cancer cells. We suppose that inner cell heterogeneity of parental MCF-7 cells is a source of observed change in CPS1 expression in MCF-7/PacR. Other mechanism affects the genes that are localized, not close to ABCB1, on the long arm of chromosome 7.
References
1. Němcová-Fürstová V, Kopperová D, Balušíková K et al.: Characterization of acquired paclitaxel resistance of breast cancer cells and involvement of ABC transporters. Toxicol Appl Pharmacol 2016; 310, 215-228.
2. Pavlíková N, Bartoňová I, Balušíková K et al.: Differentially expressed proteins in human MCF-7 breast cancer cells sensitive and resistant to paclitaxel. Exp Cell Res 2015; 333, 1-10.
3. Jelínek M, Balušíková K, Daniel P et al.: Substituents at the C3' and C3'N positions are critical for taxanes to overcome acquired resistance of cancer cells to paclitaxel. Toxicol Appl Pharmacol 2018; 347, 79-91.
4. Daniel P, Halada P, Jelínek M et al.: Differentially Expressed Mitochondrial Proteins in Human MCF7 Breast Cancer Cells Resistant to Paclitaxel. Int J Mol Sci 2019, 20, 2986.
5. Bailey PSJ, Ortmann, BM, Martinelli, AW et al.: ABHD11 maintains 2-oxoglutarate metabolism by preserving functional lipoylation of the 2-oxoglutarate dehydrogenase complex. Nat Commun 2020, 11, 4046.
Additional important information about the author Year of Graduation (Master’s degree): 2014
Year of beginning Ph.D. studies: 2014
Topic of Ph.D. dissertation: Mechanisms of taxane resistance induction in breast cancer cells
Role of the author and co-authors in preparing and carrying out the research included in the presentation:
Petr Daniel carried out 2D-PAGE, immunobloting and designed the experiments.
Petr Halada carried out MALDI-TOF analysis.
Michael Jelínek carried out confocal microscopy.
Jan Kovar supervised the project.
Prof. Iwao Ojima (Sony Brooke University, New York) synthetized and kindly provided Stony Brooke Taxane 0035.
A NOVEL ROLE FOR FIBCD1 IN THE BRAIN Christopher W. Fell
1,2,3*Co-authors: Astrid Hagelkrüys
4*, Ana Cicvaric
5,6, Johannes Stadlmann
4, Stefan Mereiter
4, Joshua Li
7, Lucy Liu
7, Marion Horrer
4, Francisco Quiroga Monje
5, Norbert Perrimon
8, Josef
M Penninger
4#and Vanja Nagy
1,2,3#1 – Ludwig Boltzmann Institute for Rare and Undiagnosed Disease (LBI-RUD), Vienna, Austria
2 – Research Centre for Molecular Medicine (CeMM), Vienna, Austria
3 – Dept. of Neurology, Medical University of Vienna (MUW), Vienna, Austria 4 – Institute for Molecular Biotechnology (IMBA), Vienna, Austria
5 – Centre for Physiology and Pharmacology, MUW, Vienna, Austria
6 – Dept. of Psychiatry and Behavioral Sciences, Feinberg School of Medicine, Northwestern University, Chicago, USA
7 – Dept. of Genetics, Harvard Medical School, Boston, USA
8 – Dept. of Genetics, Harvard Medical School, Howard Hughes Medical Institute, Boston, USA
* contributed equally
# co-corresponding Tutor: Vanja Nagy, PhD
Fibrinogen-like Recognition Domain Containing 1 (FIBCD1) encodes a single-pass type II transmembrane lectin protein with a predicted endocytic function (Shrive et al., 2014). FIBCD1 is a poorly studied gene with no link to neuroscience published thus far. Previous work has shown a role for FIBCD1 in the recognition of chitin component of fungal cell walls and a role in innate immune response to fungal infection (Schlosser et al., 2009;
Jespen et al., 2018). However, publicly available datasets and our own data show FIBCD1 to be highly expressed in human and mouse hippocampus and, in mice, FIBCD1 expression is largely restricted to neurons.
Why is a chitin-binding receptor expressed in the hippocampus? The brain’s Extracellular Matrix (ECM) is partially composed of Chondroitin Sulphate Proteoglycans (CSPGs), which have a similar polysaccharide structure to chitin, and FIBCD1 was predicted to be equally as likely to bind chitin as CSPG (Shrive et al., 2014).
The recognition and response to the CSPG ‘sulphation code’ of the brain’s ECM is key to proper development and function (Smith et al., 2015).
In the present study, we posit that FIBCD1 is a novel endocytic receptor to Chondroitin Sulphate-4S (CS- 4S). In Fibcd1-KO mouse hippocampi, we show by HPLC and Western blot that there is an accumulation of CS- 4S in adults. Fibcd1-WT primary hippocampal neurons in vitro show perturbed morphology when plated on CSPGs (aligned with previous reports (Jin et al., 2018)), but this is not the case for Fibcd1-KO neurons, indicating that CSPG to neuron signaling is at least partially mediated by FIBCD1.
We show that Fibcd1-KO mice perform poorly in hippocampus-dependent learning tasks compared to WT and HET mice, including inhibitory avoidance. In Drosophila melanogaster, neuron-specific RNAi-mediated knockdown of Fibcd1 orthologue impairs Climbing Assay performance, a common assay for neuronal function.
Finally, electrophysiological recordings taken from mouse hippocampal slices revealed an impairment in long term potentiation (LTP) in Fibcd1-KO mice compared to WT among other synaptic re-modelling phenotypes, which may be because of CS-4S accumulation shown previously. Pre-treatment of slices with the enzyme Chondroitinase-ABC (ChABC), which digests CSPGs, impaired LTP in WT mice (previously reported).
Surprisingly, ChABC treatment of KO slices rescued LTP recordings to WT levels, likely due to the removal of the accumulated CS-4S.
In conclusion, we have shown FIBCD1 is a novel receptor for CS-4S, key to neuronal development and function in mice and Drosophila melanogaster. Ongoing work includes further elucidating the relationship between FIBCD1 and CSPGs (e.g. does FIBCD1 indeed endocytose these sugars?) and if our works translates into human physiology.
REFERENCES
1. Jepsen, C. S., et al. (2018). "FIBCD1 Binds Aspergillus fumigatus and Regulates Lung Epithelial Response to Cell Wall Components." Front Immunol 9: 1967.
2. Jin, J., et al. (2018). "Effect of chondroitin sulfate proteoglycans on neuronal cell adhesion, spreading and neurite growth in culture." Neural Regen Res 13(2): 289-297.
3. Schlosser, A., et al. (2009). "Characterization of FIBCD1 as an acetyl group-binding receptor that binds chitin." J Immunol 183(6): 3800-3809.
4. Shrive, A. K., et al. (2014). "Crystal structure of the tetrameric fibrinogen-like recognition domain of fibrinogen C domain containing 1 (FIBCD1) protein." J Biol Chem 289(5): 2880-2887.
5. Smith, P. D., et al. (2015). ""GAG-ing with the neuron": The role of glycosaminoglycan patterning in the central nervous system." Exp Neurol 274(Pt B): 100-114.
ADDITIONAL INFORMATION
Year of graduation (Master’s degree): 2017 Year of beginning Ph.D. studies: 2017
Topic of Ph.D. dissertation: Genomics of rare intellectual disabilities
Role of the author and co-authors in preparing and carrying out the research included in the presentation:
CWF, AH – coordinated the project, designed and performed molecular, cellular and animal experiments, analysed data
AC, FQM – designed and performed electrophysiology experiments JS – designed and performed HPLC experiments
SM – designed and performed FACS experiments
JL, LL, NP – designed and performed Drosophila experiments MH – technically supported the work
JMP – conceptualized the study
VN – conceptualized and coordinated the study, performed behaviour experiments
SYMPATHETIC NERVOUS SYSTEM ACTIVITY IN AUTISM SPECTRUM DISORDER – EFFECT OF POSTURE AND TREATMENT
Nikola Ferencova
Department of Physiology and Biomedical Center Martin (Biomed), Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovak Republic,
Psychiatric Clinic, Jessenius Faculty of Medicine, Comenius University in Bratislava, University Hospital Martin, Slovak Republic
Co-authors: I. Bujnakova, L. Bona Olexova, Z. Visnovcova, I. Ondrejka, I. Tonhajzerova
Tutor: I. Tonhajzerova
Introduction
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social communication and interaction, and by the presence of restrictive and repetitive behavior (1). Moreover, ASD individuals engage in problem behaviors, such as aggression that may be caused by high levels of stress (2,3).
Thus, knowing the internal physiological stress state of individuals with ASD may be helpful to reduce the occurrence of such problem behavior. In this context, psychophysiological marker to assess an individuals’ internal stress state is the measurement of electrodermal activity (EDA) (4). EDA represents a non-invasive index of sympathetic cholinergic activity – activation of sympathetic nervous system leads to secretion of sweat from eccrine sweat glands resulting in increased EDA (4). EDA measurement seems to be sensitive to abnormalities in sympathetic arousal to stress which are associated with ASD (5). In addition, ASD children often suffer from comorbid diseases such as attention deficit/hyperactivity disorder (ADHD) associated with psychotropic medication potentially influencing autonomic nervous system activity. Thus, we have focused on EDA evaluation as a sensitive marker of sympathetic activity in ASD treated vs non-treated children at rest and in response to orthostatic stress (i.e. change of posture from lying to standing) associated with the sympathetic activation and decrease of parasympathetic activity (i.e. vagal withdrawal).
Methods
We have examined 23 ASD children (3 females, average age 11.1 ± 0.5 years). The diagnosis of ASD was assessed by a child and adolescent psychiatrist according to the diagnostic criteria of DSM-5 (1). ASD children were divided into 2 subgroups: ASD non-treated group (n=12) and ASD treated group with comorbidities and pharmacological treatment (n=11). The psychopharmacological drugs (methylphenidate, atomoxetine) used in ASD treated group were indicated mainly according to comorbid ADHD-like symptoms. All participants were examined in the psychophysiological laboratory with minimization of stimuli under standard conditions (same light intensity, temperature: 22-23°C). EDA was recorded during the orthostatic maneuver (change of posture from lying to standing), lasted 5 min. Two sensors (dry Ag/AgCl electrodes) were attached to the palmar surface of the medial phalanges of the second and fourth digits of non-dominant hand. This allows measurement of skin conductance level, which vary with sweat gland activity due to stress. The parameter EDA amplitude (μS) was evaluated by using a FlexComp Infinity device (Thought Technology Ltd., Canada) and computed as an average of 5 min period.
Results
With respect to treatment, EDA was significantly lower in the orthostatic phase (p = 0.027) in the ASD non-treated group compared to the ASD treated group. Moreover, EDA analysis showed a tendency towards a significant difference between the ASD non-treated and ASD treated group (p = 0.074).
Discussion
The study revealed lower EDA amplitude indicating sympathetic underarousal in response to stress (posture change) in untreated ASD children compared to treated ones pointing to the difference in sympathetic activation depending on treatment. With respect to specific comorbid ADHD treatment, methylphenidate acts as a central stimulant inhibiting the reuptake of dopamine and norepinephrine into the presynaptic neurone and increasing the release of these monoamines into the extraneuronal space in the prefrontal cortex. Together with well documented findings of improvement in cognitive performance (6), and reduction of hyperactivity and impulsivity (7), methylphenidate also has an effect on autonomic nervous system functioning (e.g. increasing heart rate and blood pressure) (8). Moreover, methylphenidate was found to increase skin conductance level indicating increased sympathetic activity (9). From this perspective, the research pointed to potential up-regulating or normalising effect of stimulant medication on autonomic nervous system activity (reviewed by 10), however, according to divergent methodological approaches and findings in the studies, further research in this area is needed.
Regarding atomoxetine as a specific norepinephrine reuptake inhibitor is considered to increase noradrenergic neurotransmission in the prefrontal cortex and consequently influence autonomic nervous system activity in the manner of increasing heart rate and blood pressure (11). However, to the best of our knowledge, the effect on skin conductance remains unknown. In this context, future research comparing the effects of stimulants (e.g.
metylphenidate) and non-stimulants (e.g. atomoxetine) could further illuminate the precise effect on autonomic nervous system activity and may help identify individuals more responsive to one type of medication than the other.
Conclusions
Our study pointed to the sympathetic underarousal indexed by EDA in response to stress in the untreated ASD group compared to the treated ASD group indicating different sympathetic reflex control depending on treatment.
It seems that electrodermal activity could represent sensitive non-invasive marker for monitoring of treatment effect in autism.
Summary
Autonomic nervous system plays an important role in maintaining homeostasis and physiological flexibility and adaptability of the organism. Dysfunction of this system may represent a crucial role in the autism pathogenesis contributing to cognitive, behavioral, and affective abnormalities associated with autism. Measurement of electrodermal activity as a non-invasive method provides an important information related to sympathetic nervous system activity and arousal. However, studies regarding EDA in autistic children are rare. Thus, we aimed to assess EDA in ASD children at rest and in response to orthostatic maneuver. We have examined twenty-three autistic children divided into ASD non-treated group and ASD treated group. The evaluated parameter was EDA amplitude (μS). Our study revealed sympathetic nervous system underactivity in response to stress-related orthostasis in the ASD non-treated group compared to the treated ASD group. In this context, electrodermal activity could represent a sensitive non-invasive marker for monitoring of treatment effectiveness in autism.
Key words: autism spectrum disorder, autonomic nervous system, electrodermal activity, treatment.
References
1. American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 5th ed.
Washington (DC): American Psychiatric Publisher 2013; 50-59.
2. Kanne SM, Mazurek MO. Aggression in children and adolescents with ASD: Prevalence and risk factors.
J Autism Dev Disord 2011; 41, 926-937.
3. Groden J, Cautela J, Prince S, Berryman J. The impact of stress and anxiety on individuals with autism and developmental disabilities. Behav Issues Autism 1994; 177-194.
4. Ferguson BJ, Hamlin T, Lantz JF, Villavicencio T, Coles J, Beversdorf DQ. Examining the association between electrodermal activity and problem behavior in severe autism spectrum disorder: A feasibility study. Front Psychiatry 2019; 10, 654.
5. Baker JK, Fenning RM, Erath SA, Baucom BR, Moffitt J, Howland MA. Sympathetic under-arousal and externalizing behavior problems in children with autism spectrum disorder. J Abnorm Child Psychol 2018;
46, 895-906.
6. Coghill DR, Seth S, Pedroso S, Usala T, Currie J, Gagliano A. Effects of methylphenidate on cognitive functions in children and adolescents with attention-deficit/hyperactivity disorder: evidence from a systematic review and a meta-analysis. Biol Psychiatry 2014; 76, 603-615.
7. Faraone SV, Buitelaar J. Comparing the efficacy of stimulants for ADHD in children and adolescents using meta-analysis. Eur Child Adolesc Psychiatry 2010; 19, 353-364.
8. Liang EF, Lim SZ, Tam WW, Ho CS, Zhang MW, McIntyre RS, et al. The effect of methylphenidate and Atomoxetine on heart rate and systolic blood pressure in young people and adults with attention-deficit hyperactivity disorder (ADHD): systematic review, meta-analysis, and meta-regression. Int J Environ Res Public Health 2018; 15, 1789.
9. Lawrence CA, Barry RJ, Clarke AR, Johnstone CJ, McCarthy R, Selikowitz M, et al. Methylphenidate effects in attention deficit/hyperactivity disorder: electrodermal and ERP measures during a continuous performance task. Psychopharmacology 2005; 183, 81-91.
10. Bellato A, Arora I, Hollis C, Groom MJ. Is autonomic nervous system function atypical in attention deficit hyperactivity disorder (ADHD)? A systematic review of the evidence. Neurosci Biobehav Res 2020; 108, 182-206.
11. Awudu GA, Besag FM. Cardiovascular effects of methylphenidate, amphetamines, and atomoxetine in the treatement of attention-deficit hyperactivity disorder: an update. Drug Saf 2014; 37, 661-676.
Additional important information about the author Year of Graduation: 2015
Year of beginning Ph.D. studies: 2017
Topic of Ph.D. dissertation: Changes of the central autonomic regulation in major depressive disorder at adolescent age
Role of the author and co-authors in preparing and carrying out the research included in the presentation:
Nikola Ferencova: postgraduate student in normal and pathological physiology. She wrote all parts of the presentation and participated in the clinic evaluation of examined subjects.
Ingrid Tonhajzerova: Professor in normal and pathological physiology. She is responsible for the complex study as a tutor and contributed to the preparation of the presentation, supervision and the final approval.
Iveta Bujnakova, Lucia Bona Olexova, Zuzana Visnovcova: young researchers in the field of normal and pathological physiology. They significantly contributed to the clinic evaluation of examined participants.
Igor Ondrejka: Associate Professor, specialist in child, adolescent and adult psychiatry. He significantly contributed to the final approval of the study.
