ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o.

ABSTRACTS – ORAL PRESENTATIONS 

14. FLOW CYTOMETRY AND PLANT GENOMICS: A HAPPY MARRIAGE 

Jaroslav Doležel, Hana Šimková, Jan Šafář, Jan Vrána, Petr Cápal, David Kopecký, 

Veronika Burešová, Jarmila Číhalíková, Marie Kubaláková, Jan Bartoš 

Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of 

Experimental Botany AS CR, Šlechtitelů 31, CZ-78371 Olomouc-Holice, Czech Republic; 

Email: dolezel@ueb.cas.cz 

The use of flow cytometry in plant science has been largely limited to the analysis 

of ploidy, nuclear genome size, and cell cycle. Other applications are less frequent, 

probably due to difficulties with preparation of samples suitable for flow cytometry from 

plant tissues and cells with rigid cell walls. However, one application has been gaining 

a growing attention and that is the chromosome sorting, which turned out to be very 

useful in plant genome analysis. Genomics aims to sequence and analyze the structure 

and function of complete genomes. However, this may be difficult in a number of species, 

including important agricultural crops, whose genomes are large and composed mostly of 

repetitive DNA. Moreover, many species are recent polyploids and interspecific hybrids. 

Sequence redundancy and presence of homologous and homoeologous sequences 

hampers gene mapping and cloning, construction of physical maps and sequence 

assembly. These tasks can be simplified after reducing sample complexity by dissecting 

nuclear genomes to single chromosomes and chromosome arms. We have shown 

previously that it is possible to prepare suspensions of intact mitotic chromosomes from 

synchronized root tips. Following this, we have developed a portfolio of applications of 

flow-sorted plant chromosomes, that includes physical mapping using PCR and FISH, 

construction of BAC libraries to facilitate construction of physical maps, and targeted 

development of molecular markers to saturate genetic maps. The International Wheat 

Genome Sequencing Consortium choose chromosome based strategy to develop ready 

to sequence physical map of hexaploid bread wheat, whose genome is almost 6-fold 

larger than that of human. The advent of next generation sequencing technology opened 

avenues for rapid sequencing DNA of isolated chromosomes, providing easy access to 

DNA composition of chromosomes. For example, this approach enabled identification 

of most of genes in barley and establishment of their order along the seven barley 

chromosomes. Sequencing flow-sorted rye supernumerary B chromosomes clarified their 

molecular structure and evolution from rye A chromosomes. Until recently, the potential 

of chromosome sorting was limited by the inability to discriminate chromosomes 

with the same DNA amount. A solution was to use special cytogenetic stocks such as 

chromosome deletion, addition or translocation lines, which are not readily available 

in many species. The constraint was overcome after Giorgi and colleagues developed 

a method called FISHIS for fluorescent labeling of DNA sequences on chromosomes in 

suspension (PLoS ONE 8: e57994, 2013). Importantly, this method provides opportunity 

46 Analytical Cytometry VII

to employ flow cytometry for estimation of the number of particular DNA sequences on 

chromosomes of interest. The numerous and important contributions to the analysis 

of complex plant genomes document how fruitful and happy the marriage between 

flow cytometry and plant genomics has been. Considering the recent methodological 

advances, one may expect that this relationship will flourish even more than before. 

18. THE PLANAR CELL POLARITY PATHWAY DRIVES PATHOGENESIS OF CHRONIC 

LYMPHOCYTIC LEUKEMIA BY THE REGULATION OF B-LYMPHOCYTE MIGRATION 

Markéta Kaucká 1 , Šárka Pavlová 2,3 , Pavlína Janovská 1 , Karla Plevová 2,3 , Lucie Poppová 2,3 , 

Hana Mádrová 2,3 , Jan Verner 2 , Jiřina Procházková 1 , Šárka Pospíšilová 2,3 , Alois Kozubík 1,4 , 

Gunnar Schulte 1,5 and Vítězslav Bryja 1,4 

1 

Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 

611 37, Brno, Czech Republic; bryja@sci.muni.cz 

2 

Center of Molecular Biology and Gene Therapy, Department of Internal Medicine– 

Hematology and Oncology, University Hospital Brno and Medical Faculty MU, Brno, 

Czech Republic 

3 

CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech 

Republic 

4 

Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech 

Republic, Brno, Czech Republic 

5 

Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden 

Chronic Lymphocytic Leukemia (CLL) is the most common form of leukemia in 

Western countries found in adults. CLL is characterized by monoclonal expansion and 

accumulation of functionally incompetent B lymphocytes. Most patients are diagnosed 

with no symptoms using a routine blood test that shows a high white blood cell count. 

CD5 + lymphocytes show impaired migration and often invade into such organs as 

lymph nodes, spleen, liver and bone marrow which results in disrupted organ function, 

hematopoiesis (anemia) and weaken immunity. The prognosis of patients with CLL varies 

widely at diagnosis and the pathology of CLL remains still unclear. 

In our study, we show that core components of Wnt/planar cell polarity (PCP) pathway 

are upregulated in B lymphocytes from patients suffering from CLL. PCP is highly 

conserved signaling machinery, which acts as key regulator of cell polarity and migration 

throughout evolution. Several players of the PCP pathway, such as Prickle1, Celsr1, 

Vangl2, casein kinase 1 (Ck1ε), Dvl2, Dvl3, Fzd3, Fzd7 and Wnt5a, are increased in CLL 

cells both on mRNA and protein levels. The expression levels of PCP genes and proteins 

often correlate with the aggressiveness of the disease and some of the PCP genes have 

prognostic value. 

Further, our analysis of the migratory capacity of CLL model cell line Mec1 and human 

primary CLL lymphocytes in the chemokine gradient (Transwell Assay) suggests that 

PCP pathway is required for migration of leukemic cells. We were able to decrease 

CLL lymphocyte migration using (i) Ror1 blocking antibody, (ii) CK1 inhibitor D4476 

Analytical Cytometry VII 47

and (iii) siRNA-mediated knockdown of Dvl2. These findings were confirmed by the in 

vivo analysis of CLL homing (using human primary CLL lymphocytes transplantations 

into immunodeficient NOD SCID gammaC-/- mice) following treatment with the Ror1 

blocking antibody and CK1-specific inhibitors. Moreover, using confocal microscopy 

and live cell imaging we demonstrate that PCP proteins are polarized in migrating Mec1 

cells in CCL19 chemokine gradient. Mec1 cells treated with CK1 inhibitor show impaired 

chemokine-driven migration in the Dunn Chamber system in comparison to untreated 

cells. 

In summary, our data demonstrate that Wnt/PCP pathway acts as the key regulator of 

impaired CLL migration and can serve as a potential target for CLL therapy. 

Acknowledgements 

This work was supported by grants from the Czech Science Foundation (301/11/0747), 

Ministry of Health of the Czech Republic (NT11217-5/2010, NS10439-3/2009, 

FNBr 65269705), Masaryk University (MUNI/A/0723/2012) and European Regional 

Development Fund (CZ.1.07/2.3.00/20.0180, CZ.1.05/1.1.00/02.0068). 

24. ANALYSIS OF DNA REPAIR FOCI BY FLOW CYTOMETRY AND MICROSCOPY AFTER 

IRRADIATION TO LOW-DOSE IONIZING RADIATION 

Matus Durdik 1 , Jan Gursky 1 , Lenka Vokalova 1 , Miroslav Kubes 2 , Eva Markova 1 , Igor 

Belyaev 1 

1 

Cancer Research Institute, Bratislava, Slovak Republic; matusdurdik17@gmail.com 

2 

Eurocord-Slovakia, Bratislava, Slovak Republic 

It is known that DNA double strand breaks (DSB) is the most severe DNA damage involved 

in many effects of ionizing radiation and other genotoxic factors. Nowadays, the most 

common method for DSB analysis is enumeration of DNA repair foci, which are formed 

at the locations of DSB. Phosphorylated histone H2AX variant (γH2AX) and p53 binding 

protein 1 (53BP1) are established as appropriate molecular markers for DSB (Bekker- 

Jensen et al., 2006). 

Emerging evidence suggests that irradiation in low doses increases cancer risks 

(Wakeford, 2013). Exposure to low doses of ionizing radiation in the range of 1 mGy 

-5 cGy commonly occurs at medical examinations, like RTG or CT, in airplanes during 

ordinary flights, or at security controls. Radiosensitive subjects, which represent 

approximately 5% of patients irradiated with therapeutical doses of ionizing radiations, 

may be especially sensitive to low doses (Goodarzi and Jeggo, 2012). In different studies, 

it was reported that doses about 1cGy induce DSB measured with DNA repair foci 

(Belyaev, 2010). The main problem in analysis of low-dose effects is poor reproducibility 

of data in various laboratories and lack of sensitive standardized methods. In previous 

experiments, analysis of γH2AX by flow cytometry was not as sensitive as microscopic 

analysis. 

We analyzed ionizing radiation induced foci (IRIF) foci, 53BP1 and γH2AX, foci induced by 

low dose gamma-rays (2, 5, 10, 20, 50cGy) in lymphocytes from umbilical cord blood using 


two sophisticated systems: Metafer (Metasystems, Germany) and ImageStream (Amnis, 

USA). Metafer is automatized microscopic system that has recently been introduced 

for analyzing DNA repair foci. ImageStream is a combination of flow cytometer with 

fluorescent microscope. Thus, we compare ImageStream with Metafer in analyzing 

IRIF. We found that sensitivity of both systems to low-dose-induced γH2AX foci was 

very similar: ImageStream show statistical significant induction at the dose of 5cGy and 

Metafer at the dose of 10cGy while prior analysis using classical flow cytometry showed 

statistical significance at doses ≥ 50cGy. Analysis of 53BP1 foci by ImageStream was less 

sensitive then by Metafer. 

In conclusion, we show for the first time that analysis of low-dose-induced γH2AX foci by 

ImageStream system is very appropriate method, that reach sensitivity of automatized 

Metafer microscopic system and is more sensitive than measuring γH2AX fluorescence 

by classical flow cytometry. Thus, ImageStream is the system that has great potential for 

analyzing DNA repair foci. 


This work was supported by the Slovak Research and Development Agency (APVV 0669- 

10) and the VEGA Grant Agency (2/0150/11) of the Slovak Republic. 

References 

Bekker-Jensen, S., Lukas, C., Kitagawa, R., Melander, F., Kastan, M. B., Bartek, J. and Lukas, 

J.: Spatial organization of the mammalian genome surveillance machinery in response to 

DNA strand breaks. - Journal of Cell Biology 173, 195-206, 2006. 

Belyaev, I. Y.: Radiation-induced DNA repair foci: Spatio-temporal aspects of formation, 

application for assessment of radiosensitivity and biological dosimetry. - Mutation 

Research-Reviews in Mutation Research 704, 132-141, 2010. 

Goodarzi, A. A. and Jeggo, P. A.: Irradiation induced foci (IRIF) as a biomarker for 

radiosensitivity. - Mutat Res 736, 39-47, 2012. 

Wakeford, R.: The risk of childhood leukaemia following exposure to ionising radiation--a 

review. - J Radiol Prot 33, 1-25, 2013. 

Legend to figure 

Typical data obtained by ImageStream 

after irradiation with the dose of 50cGy 

(transformed in black and white). 

There are 6 channels shown, from the 

left: brightfield, γH2AX stained with 

monoclonal mouse antibody and FITC., 

granularity, DNA stained with DAPI, 53BP1 

stained with polyclonal rabbit antibody 

and AF647 and colocalization of γH2AX 

and 53BP1. 


25. PROFILING OF CHILDHOOD ACUTE LEUKAEMIA CELLS USING A NOVEL FLOW 

CYTOMETRY-BASED METHOD OF AFFINITY PROTEOMICS 

Daniela Černá 1 , Veronika Kanderová 1 , Jan Stuchlý 1 , Karel Fišer 1 , WeiWei Wu 2 , Anders 

Holm 2 , Ondřej Hrušák 1 , Fridtjof Lund-Johansen 2 , Tomáš Kalina 1 

1 

Pediatric Hematology and Oncology, Charles University Prague, 2 nd Medical Faculty, 

Praha 5, Czech Republic; cerna.daniela@lfmotol.cuni.cz 

2 

Dpt. of Haematology, Rikshospitalet, Oslo, 02770, Norway 

Acute leukaemia (AL) is the most common childhood malignancy driven by a number 

of aberrations at the DNA and mRNA level. Current research is mainly focused on 

the detection of mutations at the DNA level while functional consequences of these 

alterations on cellular level are not fully understood. Proteins are entities that form 

connection between gene expression and cell physiology, therefore more effective and 

sensitive approaches that could search for new prognostic markers on protein level are 

in development. 

Using SEC-MAP technology (Size-exclusion Chromatography - Microsphere-based Affinity 

Proteomics), we are searching for the expression and activation (e.g. phosphorylation) of 

differentially expressed proteins in AL cells regarding to cell type (B-cell, T-cell, myeloid), 

genotype (fusion genes, aneuploidy) and early response to treatment detected as 

minimal residual disease. 

SEC-MAP is a set of 1728 populations of fluorescently-labeled latex microbeads, each 

carrying an antibody against a human protein. We isolate the cellular proteins from 

membranes, cytoplasm and nuclei using detergents, label them with biotin and separate 

them using gel chromatography into 24 fractions. These fractions are incubated with 

SEC-MAP microbeads and the antibody-protein binding is detected using fluorescentlylabeled 

streptavidin by flow cytometry. 

We have examined the expression of cytoplasmic (n=980) and membrane (plus DNAbinding) 

(n=769) proteins in 69 diagnostic samples of AL. The analysis was performed 

using automatic software created in R-project. For the normalization of protein 

expression we have used Loess normalization commonly used in mRNA profiling studies. 

Due to ability of SEC-MAP to separate proteins according their molecular weight we have 

identified not only the expression of particular proteins but also the size that could serve 

as a control of proteolysis. We have detected proteolysis in 12 samples, which have been 

therefore excluded from the analysis. We have revealed the sensitivity to proteolysis of 

four standard house-keeping proteins (Akt, Abl, β-actin and β2-microglobulin). Abl and 

Akt have been cleaved while β-actin and β2-microglobulin have not been detected in 

their cleaved forms in the diagnostic AL samples. Therefore we have decided to use Abl 

and Akt as house-keeping proteins to reveal the proteolysis. So far we have identified 44 

proteins (e.g. SH2D1A, FAS, LAT, KIT, CD72) differentially expressed in different subtypes 

of AL. We have discovered e.g. higher expression of cAMP-dependent protein kinase 

PRKACA in TEL-AML1 positive AL. Recently we are verifying SEC-MAP data using other 

proteomic approaches. 

SEC-MAP is a novel method of functional proteomics combining the capacity of DNA 


microarrays and high-throughput evaluation by flow cytometry, while detecting also 

changes in posttranslational modification and cellular localization. 


This work was supported by GAUK 596912, IGA NT13462, P302/12/G101, UNCE 204012, 

00064203, IGA NT12397. 

26. THE EFFECTS OF POTASSIUM CHANNEL INHIBITION ON CALCIUM INFLUX OF 

PERIPHERAL T LYMPHOCYTES IN RHEUMATOID ARTHRITIS VERIFIED USING KINETIC 

FLOW CYTOMETRY ANALYSIS 

Gergely Toldi 1 *, Anna Bajnok 1 , Diána Dobi 2 , Ambrus Kaposi 1 , László Kovács 2 , Barna 

Vásárhelyi 3 , Attila Balog 2 

*toldigergely@yahoo.com 

1 

First Department of Pediatrics, Semmelweis University, Budapest, Hungary 

2 

Department of Rheumatology, University of Szeged, Szeged, Hungary 

3 

Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary 

Background: The transient increase of the cytoplasmic free calcium level plays a key 

role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are 

important regulators of the maintenance of calcium influx during lymphocyte activation 

and present a possible target for selective immunomodulation. We aimed to characterize 

the effects of lymphocyte potassium channel inhibition on peripheral blood T lymphocyte 

activation in rheumatoid arthritis (RA) compared to healthy individuals. 

Methods: We isolated peripheral lymphocytes from 10 healthy controls and 9 recently 

diagnosed RA patients receiving no anti-rheumatic treatment. We evaluated calcium 

influx kinetics following activation in CD4, Th1, Th2 and CD8 cells. We also assessed the 

sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium 

channels. 

Cells were stained with intracellular calcium binding dyes (Fluo-3 and Fura Red) and flow 

cytometry analysis was performed in a kinetic manner (BD FACSAria). Data acquired from 

the measurements were evaluated using a novel algorithm based on the calculation of 

a double-logistic function for each recording (www.facskin.com). Specific parameter 

values describing each function were also calculated and used to compare individual 

measurements in an objective manner. 

Results: The peak of calcium influx in lymphocytes isolated from RA patients is reached 

more rapidly, indicating that they respond more quickly to stimulation compared to 

controls. In healthy individuals, the inhibition of the IKCa1 channel decreased calcium 

influx in Th2 and CD4 cells to a lower extent than in Th1 and CD8 cells. On the contrary, 

the inhibition of Kv1.3 channels resulted in a larger decrease of calcium entry in Th2 and 

CD4 than in Th1 and CD8 cells. No difference was detected between Th1 and Th2 or CD4 

and CD8 cells in the sensitivity to IKCa1 channel inhibition among lymphocytes of RA 

patients. However, specific inhibition of the Kv1.3 channel acts differentially on calcium 

influx kinetics in RA lymphocyte subsets. Th2 and particularly CD8 cells are inhibited 


more dominantly than Th1 and CD4 cells. 

Conclusions: Inhibition of Kv1.3 channels does not seem to be specific enough in 

peripheral RA lymphocytes, since anti-inflammatory Th2 cells are also affected to a 

noteworthy extent. Further studies are needed to evaluate the therapeutic potential of 

lymphocyte potassium channel inhibition in RA. 

27. FLOW CYTOMETRY-BASED GENETIC SCREEN IDENTIFIES TCF3/E2A AND TRIAP1 AS 

PATHWAY-SPECIFIC REGULATORS OF THE CELLULAR RESPONSE TO P53 ACTIVATION 

Zdenek Andrysik 1 , Jihye Kim 2 , Aik Choon Tan 2 and Joaquín M. Espinosa 1 

1 

Howard Hughes Medical Institute & Department of Molecular, Cellular and 

Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309, 

U.S.A. 

2 

Department of Medicine/Medical Oncology, University of Colorado at Denver Anschutz 

Medical Campus, Aurora, Colorado 80045, U.S.A. 

SUMMARY 

The p53 transcription factor participates in diverse cellular responses to stress including 

cell cycle arrest, apoptosis, senescence and autophagy. The molecular mechanisms 

defining the ultimate outcome to p53 activation remain poorly characterized. We 

developed a protocol for flow cytometric detection of the p53 target genes CDKN1A 

(p21), an inhibitor of cell cycle progression, versus BBC3 (PUMA), a key mediator of 

apoptosis and performed a genome wide genetic screen to identify pathway-specific 

co-regulators p21 and PUMA. Using genome-wide shRNA library our screen identified 

numerous factors whose inactivation creates an imbalance in the p21:PUMA ratio upon 

p53 activation. The transcription factor TCF3/E2A drives p21 expression while repressing 

PUMA across cancer cell types of multiple origins. Accordingly, TCF3/E2A depletion 

impairs the cell cycle arrest response and promotes apoptosis upon p53 activation 

by chemotherapeutic agents. In contrast, TRIAP1 is a specific repressor of p21 whose 

depletion slows down cell cycle progression. Our results reveal a wealth of strategies 

to drive cells into specific p53-dependent responses. Confirming cellular effects of 

candidate genes predicted by our screen we also validated an innovative approach for 

identification of co-regulators of two or more proteins. 


28. S-ADENOSYLHOMOCYSTEINE HYDROLASE REGULATES C2-CERAMIDE INDUCED 

CELL DEATH VIA INHIBITION OF THE INDUCTION OF THE INTRINSIC APOPTOTIC 

PATHWAY 

Roman Hudec 1,3,* , Kozo Hamada 1,2 , Hideaki Ando 1,2 and Katsuhiko Mikoshiba 1,2 

1 

Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 

Hirosawa, Wako-shi, Saitama 351-0198, Japan 

2 

Calcium Oscillation Project, ICORP-SORST, Japan Science and Technology Agency (JST), 

4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan 

3 

Institute of Biochemistry, Nutrition and Health Protection, Department of Biochemistry 

and Microbiology, Faculty of Chemical and Food Technology, Slovak University of 

Technology, Radlinskeho 9, 812 37 Bratislava, Slovak Republic 

*roman.hudec@stuba.sk 

S-adenosylhomocysteine hydrolase (SAHH) is the rate limited enzyme of the global 

S-adenosylmethionin (SAM) dependent transmethylation of various biological substrates. 

Here we report, that SAHH and in particular the SAHH enzymatic activity, is essential for 

the C2-ceramide induced apoptosis. SAHH knock down by the specific siRNA, disabled 

the C2-ceramide induced methylation of the catalytic subunit of protein phosphatase 2A 

(PP2Ac) and therefore its activation. Such negative regulation led to inhibition of the C2- 

ceramide induced apoptosis via mitochondrial intrinsic pathway. The opposite situation 

has been observed by the transient transfection with the plasmid carried SAHH coding 

sequence. Introduced SAHH protein caused augmentation of the endogenous SAHH 

activity and subsequently caused elevation of the C2-ceramide induced apoptosis level. 

SAHH protein levels are in general low in the neoplastic tissues; establishment or 

reestablishment of its physiological level might be beneficial for the chemotherapy 

successfulness, since many of currently used chemotherapeutics act also via mechanisms 

involving the elevation of the endogenous ceramide levels and apoptosis induction. 


This work was supported by grants from the Japan Society for the Promotion of Science 

(JSPS), the Ministry of Education, Science, Sports and Culture of Japan to K.M (20220007), 

and ICORP-SORST of JST. R.H. was a JSPS postdoctoral fellow. 


29. INFLUENCE OF THE LEVEL OF CD133 EXPRESSION ON ADHESION AND PROLIFERATION 

OF CANCER STEM-LIKE CELLS 

Radek Fedr 1# , Zuzana Pernicová 1,2# , Šárka Šimečková 1,3 , Veronika Toporcerová 1,3 , Alois 

Kozubík 1,3 , Karel Souček 1,2 * 

1 

Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the 

Czech Republic, Brno, Czech Republic; 

2 

Center of Biomolecular and Cellular Engineering, International Clinical Research 

Center, St. Anne´s University Hospital Brno, Brno, Czech Republic; 

3 

Department of Experimental Biology, Faculty of Sciences, Masaryk University, 

Brno, Czech Republic 

# 

equal contribution, *ksoucek@ibp.cz 

CD133 is a transmembrane glycoprotein that has been described as one of the typical 

cancer stem cells (CSC) marker in many types of tissue. However, the fact if the expression 

of CD133 unambiguously defines subpopulation of CSC is still controversial. Interestingly 

the function of CD133 is poorly described. Clonogenic assay is widely used in vitro for 

elucidating the proliferative capacity of every single cell in the population. Here, we 

used this assay to determine, if expression of cancer stem cell markers CD133 and 

CD44 correlates also with functional properties of cancer cells derived from conditional 

PTEN-deletion mouse model from androgen-independent tumor. Moreover, label-free 

real time cell analyzer xCELLigence was used to compare adhesion and proliferation of 

subpopulations with different expression of cancer stem cell markers. 

We found that CD44+/CD133high subpopulation of prostate cancer cells has significantly 

higher clonogenic capacity in comparison to CD44+/CD133low subpopulation. Moreover 

increased clonogenic capacity correlates with both increased adhesion and proliferation 

rate in CD133high subpopulation, as confirmed using label-free measurement with 

real time cell analyzer xCELLigence. Interestingly, fibronectin coating significantly 

increases adhesion of CD44+/CD133low and more significantly of CD44+/CD133high 

subpopulation. Blocking fibronectin motif by pre-treatment with synthetic RGDS peptide 

decreases ability of CD133high cells to adhere to fibronectin. 

We conclude that CD133 expression might contribute to the regulation of cell cycle and 

adhesion of prostate cancer cells. However further studies are necessary to describe 

mechanisms of these features. 


This work was supported by grants IGA MZD NT13573-4/2012, AV ČR M200041203, 

OrganoNET (CZ.1.07/2.4.00/31.0245), HistoPARK (CZ.1.07/2.3.00/20.0185) and 

by project FNUSA-ICRC (no. CZ.1.05/1.1.00/02.0123) from the European Regional 

Development Fund. Institutional support was provided by the Academy of Sciences of 

the Czech Republic. 


30. AN AUTOMATED ANALYSIS OF HIGHLY COMPLEX FLOW CYTOMETRY-BASED 

PROTEOMIC DATA 

Jan Stuchlý 1,2 , Veronika Kanderová 1,2 , Karel Fišer 1,2 , Daniela Černá 1,2 , Anders Holm 4 , 

WeiWei Wu 4 , Ondřej Hrušák 1,2 , Fridtjof Lund-Johansen 4 , Tomáš Kalina 1,2 

1 

Department of Pediatric Hematology and Oncology, 2 nd Faculty of Medicine, Charles 

University Prague and University Hospital Motol, Prague, Czech Republic 

2 

CLIP - Childhood Leukemia Investigation Prague 

3 

Department of Numerical Mathematics, Faculty of Mathematics and Physics, Charles 

University, Prague, Czech Republic 

4 

Department of Immunology, Rikshospitalet Medical Center and the University of Oslo, 

Oslo, Norway 

Correspondence to: jan.stuchly@lfmotol.cuni.cz 

The combination of color-coded microspheres as carriers and flow cytometry as a 

detection platform provides new opportunities for multiplexed measurement of biomolecules. 

Here, we developed a software tool capable of automated gating of colorcoded 

microspheres, automatic extraction of statistics from all subsets and validation, 

normalization, and cross-sample analysis. The approach presented in this article 

enabled us to harness the power of high-content cellular proteomics. In size exclusion 

chromatography-resolved microsphere-based affinity proteomics (Size - MAP), antibodycoupled 

microspheres are used to measure biotinylated proteins that have been 

separated by size exclusion chromatography. The captured proteins are labeled with 

streptavidin phycoerythrin and detected by multicolor flow cytometry. When the results 

from multiple size exclusion chromatography fractions are combined, binding is detected 

as discrete reactivity peaks (entities). The information obtained might be approximated 

to a multiplexed western blot. We used a microsphere set with >1000 subsets, presenting 

an approach to extract biologically relevant information. The R-project environment 

was used to sequentially recognize subsets in two-dimensional space and gate them. 

The aim was to extract the median streptavidin phycoerythrin fluorescence intensity 

for all 1000+ microsphere subsets from a series of 96 measured samples. The resulting 

text files were subjected to algorithms that identified entities across the 24 fractions. 

Thus, the original 24 data points for each antibody were compressed to 1–4 integrated 

values representing the areas of individual antibody reactivity peaks. Finally, we provide 

experimental data on cellular protein changes induced by treatment of leukemia cells 

with imatinib mesylate. The approach presented here exemplifies how large-scale flow 

cytometry data analysis can be efficiently processed to employ flow cytometry as a highcontent 

proteomics method. 


This work was supported by IGA NT/13462 and GAUK 596912 


36. B-CELL IMMUNOPHENOTYPING OF LARGE GROUP OF COMMON VARIABLE 

IMMUNODEFICIENCY PATIENTS REVEALED SUBCLUSTER WITH DISTINCT CLINICAL 

FEATURES AND SIMILAR T-CELL, CYTOKINE AND GENOMIC ABNORMALITIES 

Veronika Kanderova 1 , Jan Stuchly 1 , Marcela Vlkova 2 , Ivana Hermanova 1 , Ladislav Krol 1 , 

Marie Trkova 4 , Ondrej Hrusak 1 , Anna Sediva 3 , Jiri Litzman 2 , Eva Fronkova 1 , Tomas Kalina 1 

1 

Charles University, 2 nd Faculty of Medicine, Dpt. of Pediatric Hematology / Oncology, 

CLIP-Cytometry, Prague, Czech Republic, veronika.kanderova@lfmotol.cuni.cz 

2 

Department of Clinical Immunology and Allergology, St Anne’s University Hospital and 

Faculty of Medicine, Masaryk University, Brno, Czech Republic 

3 

Charles University, 2 nd Faculty of Medicine, Dpt. of Immunology, Prague, Czech Republic 

4 

Gennet, Genetics and Reproduction Medicine Center, Prague, Czech Republic 

Common Variable Immunodeficiency (CVID) is a heterogeneous disorder of unknown 

etiology. The hallmark of the disease is a humoral deficiency characterized by low 

levels of serum IgG, IgA, and/or IgM, impaired specific antibody response after antigen 

challenge and the resulting bacterial infections. Investigation of peripheral blood 

cellular compartments revealed abnormalities in B- and T-cells. Due to a high number of 

analysed parameters it is difficult to define CVID subgroups that possibly share the same 

pathological mechanisms. 

We have investigated detailed immunophenotype of B-cells and T-cells in CVID patients 

by 8 color flow cytometry. We have used previously reported unsupervised method of 

probability binning to compare distribution of cells within all possible phenotypes. Ninetyeight 

patients‘ and 47 healthy donors‘ samples were used to create hierarchical tree 

according to B-cell immunophenotype similarities. The cohort split into 11 phenotype 

clusters and additional one with missing B-cells. Only one B-cell cluster (further 

referenced as cluster_5) presented with characteristically aberrant immunophenotype 

of T-cells. Cluster_5 CD4+ T-cells were reduced and presented with decreased proportion 

of naive cells and increased proportion of intermediate effector memory cells (CD27- 

CD28+). Increased expression of marker of exhaustion CD57, marker of anergy PD-1 

and activation markers CD69 and CD70 suggested a chronic activation but activating 

plasma cytokine levels (e.g. IFNg, IL-2, IL-4, IL-5 measured by bead assay) were not 

elevated. Moreover, cluster_5 B-cells contained increased numbers of CD27-CD21- cells, 

low number of follicular FO II cells and reduced transitional cells as compared to other 

clusters. 

Similar clinical presentation (autoimmunity and splenomegaly) and phenotypic profile 

supported the idea of similar pathological mechanism. Whole-exome sequencing 

yielded 23 possibly damaging gene alterations (single-nucleotide polymorphisms, SNP) 

present in cluster_5 patients but not in controls. Minor allele variant of SNPs rs17615 

and rs1048971 in CR2 (CD21) gene causing alternative splicing of exon 11 was present in 

all investigated cluster_5 patients (n=6) although the frequency of minor allele in healthy 

population is 27% (p

et al. 2006), we speculate that defects in interferon induced plasma cell differentiation 

is defective in cluster_5 patients. Biological consequences of these genomic alterations 

are currently being investigated. 

The results demonstrate the usefulness of standardised flow cytometry profiling of large 

group of patients samples. 


This work was supported by IGA NT/11414-5, IGA NT/13271, P302/12/G101, UNCE 

204012, 00064203. 

References 

Asokan et. al. Characterization of Human Complement Receptor Type 2 (CR2/CD21) as 

a Receptor for IFN-α: A Potential Role in Systemic Lupus Erythematosus, The Journal of 

Immunology vol. 177 no. 1 383-394, July 1, 2006 

37. EFFECT OF GENETIC RISK FACTORS ON IMMUNE CELL PHENOTYPES IN PATIENTS 

WITH RHEUMATOID ARTHRITIS 

L. Chovanova 1,2 , M. Vlcek 1,2 , K. Krskova 1 , F. Spoutil 3 , J. Rovensky 4 , R. Brownlie 5 , 

R. Zamoyska 5 , R. Imrich 1,2 

1 

Institute of Experimental Endocrinology; lucia.chovanova@savba.sk 

2 

Center for Molecular Medicine, Slovak Academy of Sciences; 

3 

Institute of Experimental Medicine, Academy of Sciences, Prague, Czech Republic; 

4 

National Institute of Rheumatic Diseases, Piestany, Slovak Republic 

5 

Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, 

United Kingdom 

The involvement of genetics, environment and autoimmunity in the pathogenesis of 

rheumatoid arthritis (RA) has been proposed recently. The most important genetic factor 

is the shared epitope (SE) sequence in HLA-DRB1 molecule, followed by more than 30 

variants in potentially pathogenic non-MHC genes. The knowledge about their actual 

effect on immune cell function and related mechanisms is relatively poor. 

The aim of our study was to obtain a broader picture of relations between circulating 

immune cell phenotype, cytokine production after stimulation and the genetic 

background. 

PBMC were isolated from 35 healthy individuals and 36 RA patients with known genotype 

in the genes: PTPN22, STAT4, CTLA4, PADI4, AFF3, IRF5, TRAF1/C5 and HLA-DRB1. The 

proportions of selected PBMC subsets were analysed by flow cytometry. TNF-α, IFN-γ 

and IL-17 production was assessed after stimulation with PMA/ionomycin. Redundancy 

analysis (RDA) was applied to analyse the data. 

RDA analysis showed that higher proportions of memory B cells and TCRγδ cells were 

associated with the presence of risk allele in PTPN22 and AFF3. Risk alleles in IRF5, STAT4 

and TRAF1/C5 genes were associated with increased production of TNF-α and IFN-γ by 

NKT cells regardless of the diagnosis. 

Alterations in immune cell proportions and cytokine secretion are suggested as the 


possible mechanism behind risk allele association. 

Keywords: PBMC, cytokines, rheumatoid arthritis, genetics 

Acknowledgments: ATMOS N00024, VEGA 2/0018/12, ITMS 26240220058 

38. THE EFFECT OF A DIPEPTIDYL PEPTIDASE-IV INHIBITOR JANUVIA (SITAGLIPTIN) ON 

THE IMMUNE FUNCTIONS IN PATIENTS WITH TYPE 2 DIABETES 

Lucie Šromová 1 , Petr Bušek 1 , Helena Marečková 2 , Michal Anděl 3 and Aleksi Šedo 1* 

1 

Laboratory of Cancer Cell Biology, Institute of Biochemistry and Experimental 

Oncology, 1 st Faculty of Medicine, Charles University in Prague 

2 

Institute of Immunology and Microbiology, 1 st Faculty of Medicine, Charles University 

in Prague 

3 

2 nd Dept. of Internal Medicine, 3 rd Faculty of Medicine, Charles University in Prague and 

Faculty Hospital Královské Vinohrady, Czech Republic 

*Corresponding Author: aleksi@cesnet.cz 

DPP-IV is a multifunctional membrane bound serine protease identical with the marker 

of activated lymphocytes CD26 and its soluble form is found in blood plasma. Gliptins 

(e.g. sitagliptin, vildagliptin) are novel anti-diabetic drugs that inhibit the enzymatic 

activity of DPP-IV, increase the bioavailability of incretins and thus facilitate insulin 

secretion. In addition to incretins, DPP-IV may be involved in the breakdown of several 

other biologically active peptides, such as neuropeptides and chemokines. Given that 

DPP-IV has diverse effects on the modulation of cell growth and immune functions, its 

long-term inhibition could lead to unfavorable effects including immune dysregulation 

(Stulc and Sedo 2010). 

The aim of this study is to assess the possible differences in lymphocyte differentiation 

and cytokine production in patients with type 2 diabetes mellitus treated with Januvia 

(sitagliptin). 

Patients are examined before and 4 weeks after the enrolment in the study. The DPP-IV 

enzymatic activity in heparinized blood plasma is measured to confirm its inhibition in 

the treatment group. Immunophenotypisation of Treg (CD4+CD25+FoxP3+CD127-) and 

T cells (CD4+, CD8+) is performed in freshly collected peripheral blood, and Th1 (CD4+Tbet+IL-12Rbeta2+IFNgamma+), 

Th2 (CD4+STAT6+IL-4R+IL4+), Th17 (CD4+IL-17+IL-22+IL- 

23R+) populations and the presence and activation of NK cells (CD3-CD56+ lymphocytes) 

are analyzed after stimulation with phytohemaglutinine, phorbol myristate 13-acetate 

and ionomycine in the presence of brefeldin A. 

Our data show that short term (4 week) sitagliptin treatment may influence the 

proportion of lymphocyte populations in the peripheral blood. A further follow-up 

examination is planned after one year of sitagliptin treatement. This study should 

improve our understanding of the possible immunological implications of the long-term 

inhibition of the DPP-IV enzymatic activity. 


This work was supported by IGA 12407-4/2011, PRVOUK-P27/LF1/1, SVV-266 516 and 


UNCE 204013. 

References 

Stulc T., Sedo A.: Inhibition of multifunctional dipeptidyl peptidase-IV: Is there a risk of 

oncological and immunological adverse effects? Diabetes Res Clin Pract. 88(2): 125-3, 

2010 

40. NONCLASSICAL CD16 + PERIPHERAL BLOOD MONOCYTES EXPRESS CD11C WITH 

HIGH INTENSITY IN EARLY RHEUMATOID ARTHRITIS 

Olga Kryštůfková, Heřman Mann, Hana Hulejová, Ladislav Šenolt, Jiří Vencovský 

Institute of Rheumatology, Prague, Czech Republic and Dept. of Rheumatology, 

1 st Faculty of Medicine, Charles University, Prague, Czech Republic, krys@revma.cz 

Three populations of human monocytes have been described based on the relative 

expression of surface markers CD14 and CD16, as classical (CM; CD14 + CD16 - ), nonclassical 

(NCM; CD14 dim CD16 ++ ) and intermediate (IM; CD14 + CD16 +/++ ) [1]. NCM are 

potent antigen presenting cells and producers of proinflammatory cytokines, with high 

migratory ability and are involved in Fc mediated killing. They were enriched in peripheral 

blood (PB) of patients with rheumatoid arthritis (RA) and correlated with disease activity 

and titers of rheumatoid factor [2-5]. Response to therapy with methotrexate (MTX) 

was associated with reduction of CD16 expression [4,5]. Higher expression of CD16 on 

RA synovial fluid monocytes compared to PB and in RA synovium was reported. CD11c 

expressed by human monocytes, is involved in clearance of immune complexes, plays 

a role in antigen presentation and in production of proinflammatory cytokines. Higher 

expression of CD11c on mononuclear blood cells (PBMC) in patients with RA, expansion 

of CD11c positive inflammatory macrophages in RA synovium and a predictive role of 

higher CD11c mRNA expression for response to TNFα blocking treatment in RA were 

shown [6-8]. 

The aim of this study was to evaluate the expression of CD11c on monocyte 

subpopulations in patients with early rheumatoid arthritis (ERA) and to compare them 

to patients with osteoarthritis (OA). We also studied changes of CD11c expression after 

three months of treatment. 

Thirty one patients with Early Rheumatoid Arthritis (ERA) and 9 gender and age-matched 

patients with osteoarthritis (OA) were included. Patients in the ERA cohort had symptom 

duration < 6 months and either fulfilled the 2010 ACR/EULAR classification criteria for RA 

or had undifferentiated inflammatory arthritis. Serum CRP and anti-CCP autoantibody 

levels and rheumatoid factor positivity were recorded. Disease activity was assessed 

using DAS28. 

Monocytes were selected using a sequential gating approach by exclusion of doublets 

(high FSc Area plotted in versus FSc Lin), granulocytes (SSc high granular events), CD45 - 

events and HLADR - /CD3 + /CD19 + lymphocytes. Their subpopulations were defined 

based on the positivity of CD14 and CD16 according to IUIS nomenclature [1]. Classical 

monocytes were defined as CD14 + CD16 - and non-classical as CD14 dim CD16 ++ . With use of 


isotype controls and FMO for CD14 and CD16; two intermediate (IM) populations were 

evaluated separately as CD14 + CD16 + and CD14 + CD16 ++ . In these monocyte populations, 

intensity of CD11c expression was analysed as the median intensity of fluorescence 

(MFI). 

ERA patients had lower proportions of intermediate monocyte subsets (IM-CD16 + p=0.02 

and IM-CD16 ++ p=0.15) and more classical monocytes (p=0.009) compared to OA. The 

presence of thyroiditis (n=3) was associated with higher proportion of non-classical and 

intermediate monocytes (p=0.01 and p=0.04). 

Majority of monocytes expressed CD11c (median 98.2%). NC monocytes had highest 

expression of CD11c (MFI 558.9 ± 117) compared to CM (MFI 331.7 ± 93.13; p

al., Adhesion molecule expression on peripheral blood mononuclear cells in rheumatoid 

arthritis: positive correlation between the proportion of L-selectin and disease activity. 

Clin Rheumatol, 1995. 14(3): p. 335-41. 

7. Tanaka, M., et al., Expansion of a unique macrophage subset in rheumatoid arthritis 

synovial lining layer. Clin Exp Immunol, 2008. 154(1): p. 38-47. 

8. Stuhlmuller, B., et al., CD11c as a transcriptional biomarker to predict response to 

anti-TNF monotherapy with adalimumab in patients with rheumatoid arthritis. Clin 

Pharmacol Ther, 2010. 87(3): p. 311-21. 

41. IMMUNOPHENOTYPE ANALYSIS OF NUCLEATED ERYTHROID PROGENITORS. 

APPLICATION OF OBTAINED RESULTS IN LEUKAEMIA DIAGNOSTICS 

Michaela Fajtová 1,2 , Anna Kovariková 1,2 , Jan Sedlák 1 

1 

Cancer Research Institute 

2 

Centre for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovak 

Republic; e-mail: michaela.fajtova@savba.sk 

Introduction: Nucleated erythroid progenitors (NEPs) in normal regenerating bone 

marrow (BM) comprised of 4 developmental stages – pro-erythroblasts, basophilic 

erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts. 

In clinical laboratories, the erythroid phenotype is conventionally characterised by the 

expression of CD36, CD71, and CD235a antigens, however the analysis of these antigens 

is not sufficient for the distinction of NEPs into 4 developmental stages. We supplemented 

previous NEP phenotype analysis (Wood, 2004) with the analysis of additional markers 

on NEPs, including CD105, CD117, CD45, CD38, we proposed NEPs gating strategy and 

determined the percentage of NEPs developmental stages in regenerating BM (Fajtova 

et al., 2013). The knowledge of exact erythroid phenotype is helpful for uncovering 

neoplastic erythroid population in diagnosis and follow-up of acute erythroid leukaemia 

(AML-M6) and myelodysplastic syndrome (MDS). 

Material and methods: NEPs phenotype was analysed on normal BM samples from 6 

non-leukemic and 14 leukemic patients (mainly B-ALL) in complete remission. Aberrant 

NEPs phenotype was analysed on BM samples from AML-M6 patients in diagnosis and 

follow-up. Flow cytometric measurement was performed on a BDCantoII Flow Cytometer 

using 6-colour staining. NEPs phenotype profile was examined with antibodies: anti- 

CD36-FITC, anti-CD36-FITC/CD235a-PE, anti-CD71-PE, anti-CD105-PerCP-Cy5.5, anti- 

CD34-PE-Cy7, anti-CD117-APC, anti-HLA-DR-FITC, anti-CD38-PE, anti-CD45-VioGreen 

and nucleic dye Syto. 

Results: All NEP developmental stages were gated as cells with the highest CD71 intensity 

and were stained with the nucleic acid dye Syto. Alternatively, NEPs in all stages could 

be also gated as CD36 + CD45 -/low Syto + cells, as CD45 -/low gating excluded the monocytic 

population and Syto + gating excluded the denucleated erythro debris (CD36 + Syto - ). 

CD36 + CD45 -/low Syto + gating is necessary in samples lacking CD71 antigen expression on 

NEP population. 


NEP developmental stages were distinguished on the basis of different values of CD105, 

CD117, FSC parameters. Pro-erythroblasts were characterized by the expression of 

CD71 + , CD105 + , CD117 + and the highest value of FSC; basophilic erythroblasts by the 

expression of CD71 + , CD105 + and decreased FSC; polychromatophilic erythroblasts by 

the expression of CD71 + and medium FSC; orthochromatophilic erythroblasts by the 

CD71 + and the lowest FSC value. NEP developmental stages expressed CD36, CD38 and 

CD45 in various intensity and did not express CD34 and HLA-DR. 

Knowledge of the expression profiles of normal NEPs, we used for the analysis of 

erythroid population in two cases of AML-M6 subtype erythroleukaemia, which we were 

analysed in the years 2008-2013. 

In first case of erythroleukaemia we identified the presence of myeloblasts (9% from 

BM nucleated cells) with aberrant phenotype (CD7 + , CD22 + ) and NEPs (75%). NEPs 

comprised of amplified cells of different developmental stages (7% pro-erythroblasts, 

5.4% basophilic erythroblasts, 21.5% polychromatophilic erythroblasts, and 38.8% 

orthochromatophilic erythroblasts). All stages displayed aberrantly reduced CD71 

expression (decreased intensity) and orthochromatophilic erythroblast expressed 

slightly decreased intensity of CD36 (90%). 

Patient underwent treatment, achieved complete remission and subsequently 

underwent allogeneic stem cell transplantation. Nine months after diagnosis, this 

patient relapsed, showing the presence of myeloblasts (21%) and NEPs (49%) in the 

BM. Pro-erythroblasts and basophilic erythroblasts possessed normal phenotype, 

polychromatophilic erythroblasts displayed aberrantly reduced CD71 expression (50%), 

and orthochromatophilic erythroblasts lost CD71 expression (1%) and showed reduced 

CD235a expression (30%). 

In second case of erythroleukaemia we identified in diagnosis the presence of myeloblasts 

(6%) with aberrant phenotype (CD38 dim , CD15 dim , CD13 - ) and NEPs (82%) in the BM. 

Neoplastic NEPs comprised of mass of cells with aberrant phenotype (CD36 neg-bright , 

CD71 medium , CD235a + , CD105 dim , HLA-DR dim , CD38 dim a CD117 - ), aberrantly increased SSC 

characteristic and without the possibility to distinguish different NEPs developmental 

stages. Detection of minimal residual disease by flow cytometry on day 33 and 64 was 

based on identification of residual NEPs with aberrant phenotype. However, during 

follow-up we identified NEPs (2%), in which it was possible to identify all 4 developmental 

stages with normal phenotype. 

Conclusions: AML-M6 is very rare type of AML that accounts for less than 5% of adult 

AML cases. MDS is more frequent clonal malignant disorder, in which also NEPs could 

possess phenotypic abnormalities, such as asynchronous expression of CD71 versus 

CD235a (Malcovati et al., 2005), altered distribution of nucleated red blood progenitors 

and increased numbers of CD36 -/low NEPs (Matarraz et al., 2010). In clinical laboratories, 

the phenotype of neoplastic NEPs in AML-M6 or MDS is evaluated as a one whole that 

causes losing information about aberrant phenotypes on different NEP developmental 

stages - important information for leukaemia follow-up. Our proposed NEP gating scheme 

allows for the exact assessment of phenotype of different NEP developmental stages in 

BM. Evaluation of CD71, CD235a, CD117, CD105, CD36, CD45 antigen expression is also 

recommended in standardized Euroflow panels for AML/MDS. 



This work was supported by a grants from the Slovak Grant Agency VEGA (No.2/0041/10), 

(No.2/0134/13) and Grant NFM/EEA SK0095. The authors acknowledge the physicians 

(Peter Švec, MD PhD., Halina Vargová, PhD.) from the Department of Pediatric Oncology 

of University Children’s Hospital, Bratislava, Slovakia, for submitting patient bone marrow 

samples and referrals. 

References 

Wood, B. Cytometry, 4th Edition New Developments. Elsevier Academic Press 2004; 

Methods in Cell Biology 75:559–76. 

Fajtova, M., et al. Leuk Lymphoma. 2013 Apr 23. [Epub ahead of print] 

Malcovati, L., et al. Leukemia 2005; 19:776–783. 

Matarraz, S., et al. Cytometry Part B. 2010; 78B:154–168. 

42. EFFECTS OF NATALIZUMAB ON CD4+CD25HIGH LYMPHOCYTES 

Helena Mareckova 1 , Zdenka Hruskova 1 , Lucia Skacikova 1 , Eva Havrdova 2 

1 

Inst. Immunology and Microbiology, 1 st Medical Faculty, Charles University, Prague 

2 

MS Centrum Neurology Clinic, 1 st Medical Faculty, Charles University, Prague 

Background: Natalizumab is a humanized monoclonal antibody directed against very 

late activation antigen 4 (VLA-4) and has a potent effect on disease activity in multiple 

sclerosis (MS). Blockade of VLA-4 with natalizumab may not only interfere 

with autoimmune mechanisms but also with central nervous system immune surveillance. 

Methods: Longitudinal study (follow-up between 2 and 5 years) to determine the 

effect of natalizumab on the frequency of CD4+CD25high regulatory T cells (Tregs) in 

peripheral blood (PB) and in cerebrospinal fluid (CSF). We examined 58 patients with MS 

treated with Tysabri (Biogen, Idec) at baseline and during therapy. Using flow cytometry 

with six-color imaging (Canto, BD Biosciences) following markers were assessed: CD4, 

CD8, CD25, CD28, CCR5 (BD) and CXCR3 (RD) in CSF and PB. 

Results: There was a gradual increase in circulating CD4+CD25high lymphocytes in PB 

during whole follow-up and marked increase in all patients in CSF between baseline and 

control examination. Results from CSF analysis revealed that natalizumab blocks CD4+ 

more than CD8+ T cells from transmigration 

Conclusion: The evidence for the use of natalizumab in MS from clinical trials is strong, 

but knowledge on effects on different immune cell populations is limited, especially for 

CSF. Impairment of the inhibitory function of Tregs seems to play a role in MS disease 

pathogenesis. Direct effects of natalizumab on T cell function have been proposed to 

promote some of the effects in MS, and VLA-4 blockade has been reported to modulate 

the activation state of CD4+ T cells. Thus, natalizumab may positively influence frequency 

of Tregs. 

Supported by IGA MZ NT / 13108 – 4 


50. THE PROGRESSION OF HIV INFECTION IN TERMS OF LABORATORY IMMUNOLOGY 

Alexandra Lochmanová 1 , Lenka Olbrechtová 2 , Jitka Kolčáková 2 , Alena Zjevíková 2 

1 

Institute of Public Health, Department of Immunology and Allergy, Ostrava, Czech 

Republic; alexandra.lochmanova@zuova.cz 

2 

Faculty Hospital Ostrava, Clinic of Infectious Medicine, Czech Republic 

The pathogenesis of HIV infection includes depletion of the total body CD4+ T-cell pool, 

leading to immunodeficiency. This effect is accompanied by activation of numerous 

elements of immune system. Immune activation appears to be driven by both 

homeostatic response to CD4+ T/cell depletion and an inflammatory response to HIV 

infection. 

The correlation between peripheral blood CD4+ counts and the spectrum of clinical 

manifestations of HIV disease is well defined and has been recognized for many years. 

Despite its value, the peripheral blood CD4+ count is recognized as an imperfect marker 

of HIV disease progression. In some cases, the CD4+ percentage is a better marker of 

immune competence than CD4+ count. 

Measurements of T-cell function seems to be more effective and central to understanding 

HIV disease. Three types of assays are typically used to measure T-cell function: 

cytotoxicity, proliferation and cytokine secretion. Nonproliferation-base assessments 

of immune activation include measuring expression of so-called “early” cell surface 

markers such as HLA-DR, CD38, CD69 or CD25. A proliferative state as a definitive 

indicator of activation accompanied by DNA synthesis can be assessed by 3 H-thymidine 

uptake, measurements of intracellular proteins such as Ki67 or staining with tracking 

dyes (CFSE). 

Chronic HIV infection is accompanied by permanent activation of immune system a 

persistent inflammation. The intensity of this process is associated with serious outcome 

and poor prognosis. One of the most effective cytokine in thi sproces is IL-2, which is a 

potent inducer of T-cell proliferation and participates in both induction and suppression 

of inflammatory process. 

Routine laboratory monitoring of HIV patients involves determining peripheral blood 

CD3+, CD3+ CD4+ and CD3+CD8+ absolute count and T-cell proliferation measured by 

3 

H-thymidine incorporation after stimulation with PHA. 

It was show that the degree of cell activation, resp. functional activity, clearly reflects 

the depth of immunodeficiency. Reduction of functional activity indicates impairment 

of immune competence, whose improvement can be achieved therapeutically. Longterm 

unresponsiveness suggests a definite loss of immune competence and appears 

in the terminal stage of the disease. Impaired ability of lymphocyte proliferation may 

be caused by a lack of synthesis of ribonucleotides due to defect of relevant metabolic 

pathways. However, IL-2 seems to be one of the most important components of this 

process because IL2 is essential for lymphocyte proliferation. In accordance with these 

findings correspond clinical studies which indicate that administration of recombinant 

IL2 leads to a permanent increase in the number and function of CD4 + T cells in patients 

in both early-and late-stage of HIV infection. 


Our data demonstrate that simultaneous determination of the proliferative activity of 

cells with CD4+ count seems to be more effective marker for assessing the degree of 

functional immunodeficiency monitoring HIV patients than CD4+ count itself. 

References 

Streeck H, Nixon DF. T cell immunity in acute HIV-1 infection. J Infect Dis. 2010 Oct 

15;202 Suppl 2:S302-8. 

Chattopadhyay PK, Roederer M. Good cell, bad cell: flow cytometry reveals T-cell 

subsets important in HIV disease. Cytometry A. 2010 Jul;77(7):614-22. 

Vrisekoop N, Mandl JN, Germain RN. Life and death as a T lymphocyte: from immune 

protection to HIV pathogenesis. J Biol. 2009;8(10):91. 

Boasso A, Shearer GM, Chougnet C. Immune dysregulation in human 

immunodeficiency virus infection: know it, fix it, prevent it? J Intern Med. 2009 

Jan;265(1):78-96. 

51. MYSTERIES IN THE LABORATORY OF CELLULAR IMMUNOLOGY OR 

WHAT WE CAN ENCOUNTER WHEN ANALYZING DATA FROM FLOW CYTOMETER 

Doris Vokurková 1 , Pavlína Králíčková 1 , Eva Malá 1 , Jakub Novosad 1 , Pavel Rozsíval 2 , Eva 

Pařízková 2 

vokurkovad@lfhk.cuni.cz 

1 

Institute of Clinical Immunology and Allergology, Medical Faculty and Faculty Hospital, 

Hradec Kralove, Charles University in Prague, Czech Republic 

2 

Department of Pediatrics, University Hospital, Charles University, Hradec Kralove, 


One of the advanced techniques that in recent years achieved significant technological 

development is the method of flow cytometry. It plays a vital role in the immunological 

and haematological laboratory and the number of tests for examining patients continues 

to grow. A number of methods are validated, there are commercial kits for functional 

tests, regular calibration is carried out, protocols for the methods are set, but still 

sometimes we get results that deviate from the norm for other reasons than because of 

pathological value of the result. 

For example, how to interpret normal percentage values of metabolic flare in granulocytes 

with a substantially lower MFI? How to explain a relatively good clinical condition of 

a patient who according to the results of burstest and cytoplasmatic myeloperoxidase 

should suffer from serious bacterial infections? Why, from time to time, during the 

measurement of basic lymphocyte populations there appears „unbalanced“ twoparameter 

dot-plot histograms and the compensation matrix of the given sample must 

be adjusted? How to evaluate patients with recurrent high negative control in bazotest? 

Even after several years of work with a flow cytometer we encounter very baffling cases 

that are difficult to interpret and therefore they deserve to be discussed in more detail. 


52. PRŮKAZ REGULAČNÍCH T LYMFOCYTŮ – METODICKÉ ASPEKTY 

Jitka Pohořská 1 , Jan Laštovička 2 , Vlastimil Král 1 

1 

Zdravotní ústav se sídlem v Ústí nad Labem, Centrum imunologie a mikrobiologie, Ústí 

nad Labem, jitka.pohorska@zuusti.cz 

2 

Fakultní nemocnice Motol, Ústav imunologie, 2. Lékařská fakulta UK, Praha 

Snaha o identifikaci populace buněk se „supresorovou“ aktivitou se datuje již do 

počátku 90. let minulého století. Významným průlomem byl průkaz supresivní aktivity 

populace CD4 + CD25 ++ T buněk v myším systému Sakaguchim a spol. v r. 1995. Identifikace 

regulačních T lymfocytů jako CD4 + CD25 ++ lymfocytů se v lidském systému brzy ukazovala 

jako nedostatečná a byly hledány další diferenciační/aktivační znaky T buněk pro přesnější 

identifikaci Treg. Rozvoj poznatků o úloze transkripčních faktorů a výsledky experimentů 

v myším systému znamenaly zásadní posun pro identifikaci Treg. Byl objeven diferenciační 

znak myších Treg - transkripční faktor FoxP3 s výraznou expresí v populaci CD4 + CD25 hi , 

který byl díky analogii u myší považovaný za jednoznačně určující i pro člověka. Přibližně 

ve stejné době byly prováděny experimenty s průkazem Treg in situ v nádorové tkáni, 

kde byla zaměřována pozornost na molekuly ICOS (inducibilní kostimulátor indukující 

syntézu IL10) a GITR (receptor pro TNF indukovaný glukokortikoidy, jehož exprese se 

zvyšuje u aktivovaných T lymfocytů). Pokroky ve výzkumu Treg v lidském systému utlumily 

počáteční optimismus spojovaný s FoxP3 jako jednoznačným diferenciačním znakem této 

subpopulace T lymfocytů u lidí. Exprese FoxP3 není u člověka omezena pouze na Treg – 

tento transkripční faktor není (na rozdíl od myší) markerem konečného diferenciačního 

stadia. Důležitým krokem k přesnější identifikaci Treg u člověka bylo poznání fyziologické 

funkce receptoru pro IL7 (molekula CD127, alfa řetězec IL-7R). V práci Mazzucchelliho a 

spol. (2007) byla prokázána nepřímá závislost exprese CD127 a FoxP3 u aktivovaných a 

regulačních T lymfocytů. 

Dalšími slibnými kandidáty pro průkaz tlumivé funkce Treg se jeví CD152 (indukce CTLA- 

4 u revmatoidní artritidy obnovuje supresivní kapacitu Treg). V současnosti nachází 

využití v klinické praxi sledování exprese CTLA-4/CD152 v diagnostice imunologicky 

podmíněných neplodností. Pro rozlišení přirozených a indukovaných Treg subpopulací 

byl v posledních letech zmiňován transkripční faktor Helios (odlišení přirozených a 

indukovaných Treg), jehož nevýhodou je podobně jako u FoxP3 intracelulární značení. 

V současnosti se ale objevují práce, které prokazují i přirozené Treg s negativitou Helios. 

Podobně jako CD127 je zvažován další „negativní“ marker pro Treg - extracelulární 

serinová protéza s dipeptidyl-peptidázovou aktivitou (CD26). Exprese CD26 identifikuje 

Th1 efektorovou odpověď (rozdíly v expresi mezi Treg a efektorovými T lymfocyty jsou 

stabilní). 

Komplikace v metodických přístupech mohou do značné míry souviset s aktuálně 

uznávaným konceptem „diferenciační plasticity“ populace CD4+ T lymfocytů u člověka, 

která předpokládá určitou reversibilitu diferenciačních stupňů v závislosti na cytokinovém 

prostředí a expresi transkripčních faktorů. 

Budou zmíněny základní metodické přístupy stanovení Treg a jejich vliv na získaná data, 

modifikace vhodné pro detekci Treg v rutinním provozu klinické laboratoře (porovnání 


výsledků získaných ze separovaných lymfocytů a plné krve).Hladiny regulačních T 

lymfocytů byly sledovány u různých imunopatologických stavů (infekce MTB, alergie, 

autoimunitní choroby léčené biologickou terapií, atd.). 

Reference 

Bluestone JA, Zang Q., Curr. Opin. Immunol. 2005, 17:638-642, Liu W., Putnam, AL., Xuyu 

Z., et al., JEM, 2006, 203(7), 1701-1711) 

Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, Gottlieb PA, Kapranov P, Gingeras 

TR, Fazekas de St Groth B, Clayberger C, Soper DM, Ziegler SF, Bluestone JA. CD127 

expression inversely correlates with FoxP3 and suppressive function of human CD4+ T 

reg cells. J Exp Med. 2006 Jul 10; 203(7):1701-11 

Mazzucchelli M. and Scott K. Durum, Interleukin-7 receptor expression: intelligent 

design. Nature reviews Immunology Vol 7, No 6, June 2007) 

53. THE RESULTS INTERPRETATION OF BASIC LYMPHOCYTE SUBPOPULATIONS BY THE 

VIEW OF FLOW CYTOMETRIST AND CLINICAL IMMUNOLOGIST 

Marcela Vlková, Zdenka Pikulová 

Department of Clinical Immunology and Allergology, St Anne’s University Hospital, 


marcela.vlkova@fnusa.cz 

Results of immunophenotyping of peripheral blood lymphocyte subsets may 

give important information for the diagnosis and treatment of haematological or 

immunological disorders as well as infectious diseases. For example, the number of 

CD4+T-cells are used to define the stage of the human immunodeficiency virus infection, 

the number of cytotoxic T-cells and their activation status may be an important tool for 

the determination of viral infections such as cytomegalovirus or Epstein–Barr virus. It 

is necessary to keep some basic terms of sample manipulation to be able to correctly 

interpret the results of immunophenotyping of peripheral blood lymphocyte subsets 

from flow cytometer. 

The measurement process should meet the requirements of internal quality control. 

First condition is properly collected blood: proper storage of sample, transportation 

conditions, timely delivery to the laboratory. The second condition is the right 

preparation and processing of samples for measurement. Third condition involves 

functional and properly configured flow cytometer. Fourth important requirement is a 

correctly measured sample. 

If all these conditions are met we can proceed to the actual interpretation of cytometric 

data. For this we need the determination of a range of reference values of lymphocyte 

subpopulations depending on the patient’s age. At the birth the immune system is 

functionally immature and undergoes a sequential development which is followed 

as changing leukocytes and lymphocytes levels and percentage of lymphocyte 

subpopulations. This development is genetically determinated and depends on 

subsequent stimulation by antigen. These changes necessitate age-matched reference 


values when analysing paediatric lymphocyte subsets. To present the development of 

the lymphocyte subset, the relative as well as the absolute cell count is needed. 

The other changes on the numbers and percentage proportions are depend on 

actual or long-term treatment of patient. Immunophenotyping of basic lymphocyte 

subpopulations is performed most often in patients with suspected immunodeficiency. 

The results are also affected by other diagnoses such as viral or bacterial infection, 

leukaemia or cancer. For those reasons it is necessary to interpret the flow cytometric 

immunophenotyping results of patients in different age groups with different diagnoses 

from a perspective of a cytometrist and a clinical immunologist althogether. 


This work was supported by IGA NT/11414-5, IGA NT/13271 

54. KDY POMÝŠLET NA HEMATOLOGICKOU MALIGNITU V RUTINNÍM 

IMUNOLOGICKÉM VYŠETŘENÍ PRŮTOKOVOU CYTOMETRIÍ 

Ester Mejstříková, Ondřej Hrušák 

2 nd Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech 

Republic 

Hodnocení zastoupení lymfocytárních subpopulací průtokovou cytometrií v rámci 

imunologického vyšetření je základní metodou k vyloučení závažných defektů 

buněčné imunity. Vyšetření zpravidla porovnává procentuální zastoupení jednotlivých 

lymfocytárních subpopulací s věkově definovanou normou. 

Obecně podezření na hematologickou malignitu vzniká v situaci, kdy v periferní krvi je 

zřetelná populace s netypickými optickými vlastnostmi nebo když nacházíme populaci 

s odlišnou intezitou fluorescence klíčového znaku (např. CD45, CD19, CD3, CD8, 

CD4 a jiných). Typické změny nacházíme u chronické lymfatické leukémie, leukémie 

z velkých granulárních buněk (T, resp. NK buněčného původu), akutní leukémie, 

myelodysplastického syndromu (MDS). Typické změny lze nalézt rovněž u pacientů 

s pre-maligními stavy, jako je např. monoklonální B lymfocytóza či lymfocytární varianta 

eosinofilního syndromu. Významnou aberací v periferní krvi (nikoli v kostní dřeni) svědčící 

pro přítomnost atypických buněk, je záchyt populace s nízkou granularitou a expresí 

antigenu CD45 nižší než mají nemaligní lymfocyty. Abnormální granulaci granulocytů, 

která může vypadat obdobně jako MDS, nacházíme u vzácného primárního imunodeficitu 

selektivní deficience granul na podkladě mutace v transkripčním faktoru CEBPε. 

Klíčové pro správné zhodnocení lymfocytárních subpopulací je, že jednotlivé populace 

přibližně dají dohromady 100% v rámci lymfocytů a že součet CD4 a CD8 se rovná 

procentu TCRαβ pozitivních T lymfocytů. 

Některé primární imunodeficience jsou asociované s rizikem rozvoje hematologické 

malignity, u části je typický nález v předchorobí. Známá je asociace autoimunitního 

lymfoproliferativního syndromu a lymfómu či deficience transkripčního faktoru GATA- 

2 a myeloidní malignity. Imunologické vyšetření může vést ke správné diagnóze a 

monitorování pacienta. 

Podpořeno granty UNCE 204012, GAČR P/301/10/1877, NT/12425-4, NT/14534, NT13462 


55. ACTIVITY OF ALDEHYDE-DEHYDROGENASE IN B-CELL AND PLASMA CELL SUBSETS 

OF MULTIPLE MYELOMA PATIENTS 

Pavla Vsianska 1,2,3 , Lucie Rihova 1,2 , Fedor Kryukov 1,2 , Miroslav Penka 1 , Roman Hajek 1,2 

1 

Dept. of Clinical Haematology, University Hospital Brno, Brno, Czech Republic, 

ZarbochovaP@seznam.cz 

2 

Babak Myeloma Group by Dept. of Pathological Physiology, Faculty of Medicine, 

Masaryk University, Brno, Czech Republic 

3 

Dept. of Experimental Biology, Faculty of Science, Masaryk University, Brno, 


Multiple myeloma (MM) is characterized by the presence of clonal plasma cells (PC) 

arising from malignant transformed B-cells. It is still not clear which stage of B-cell 

differentiation is responsible for the development of MM and for eventual relapse after 

treatment, so nowadays there is an effort to identify the source of myeloma-initiating 

cells. Aldehyde-dehydrogenase (ALDH) is an intracellular enzyme catalysing degradation 

of aldehydes to protect the cell against a toxic damage (Russo and Hilton, 1988). This 

enzyme is active in hematopoietic stem and progenitor cells and its increased activity 

was detected also in some types of cancer stem cells (Shenoy et al., 2012; Rappa et al., 

2013). 

The aim of this study was monitoring of ALDH activity in B-cell and plasma cell 

subpopulations in MM patients to identify potential source population of myeloma 

progenitors. 

Bone marrow (BM) of 10 newly diagnosed MM patients were analysed by 8-color flow 

cytometry. Identification of immature, transitional, naïve, memory (with/without isotype 

switch) and switched CD27 - B-cells, as well as plasmablasts and PCs (CD19 + PCs, CD19 - PCs 

and CD138 -/dim+ PCs) was performed according to expression of surface markers CD38, 

CD45, CD20, CD138, CD19, CD27 and IgM. Aldefluor assay was used to identify activity 

of ALDH in individual subsets and negative control with ALDH inhibitor was used in each 

sample. Rate of ALDH activity was assessed based on percentage of ALDH positive cells 

(%pos) and ratio of median fluorescence intensity (MFI) of ALDH and MFI of negative 

control. Statistical significance of differences in continuous variables among groups of 

patients was analysed using nonparametric Kruskal-Wallis or Mann-Whitney U test. For 

the robust analysis of continuous parameters relationship the Spearman correlation 

coefficient was adopted. 

There was found an decreasing trend of ALDH activity in B-cell development from 

immature to mature naïve B-cells, then the activity is stable until the stage of PC, where 

it increases again. Higher activity of ALDH in immature B-cells in comparison with naïve 

B-cells was found according to MFI ratio [1,42 (1,15-1,79) vs. 1,03 (0,72-1,59), p

42,0) vs. 1,5 (0,0-28), p

extrinsic type of apoptosis, associated with an increase in DR5 expression, the formation 

of DR5-containing death inducing signaling complex (DISC) and subsequent activation of 

caspase-8. Supportive evidences were generated by stable overexpression of FADD-DN 

(FAS-associating death domain-containing protein-dominant negative) or c-FLIP (cellular 

FLICE-like inhibitory protein), potent inhibitors of DISC aggregation. A similar but 

significantly delayed death signaling pathway was observed in HCT116 cells lacking p53, 

as demonstrated by less prominent specific cleavage of caspases and PARP, and reduced 

mitochondrial release of cytochrome c, compared to their p53 expressing counterparts. 

More recently, we have focused on the primary trigger point of FU and our preliminary 

data indicate that the elicited death response is emerging from transcriptional but not 

from DNA lesions in some CRC cell lines. We believe that FU indeed misincorporates into 

DNA but fails to induce a stress response. Therefore, current aims involve modulation of 

DNA-repair systems in order to further augment cell death by stimulating DNA stress in 

parallel to RNA toxicity. Interestingly, in FU-treated cells suppression of PARP activity, an 

important factor in base excision repair, specifically induces DNA damage and enhances 

apoptosis in p53 deficient HCT116 cells. This response pattern has been verified also 

in other cell systems. In conclusion, lack of p53 reduces the apoptotic response to FU 

alone but allows for sensitivity to combinatorial treatment including a PARP inhibitor. 

Ongoing experiments are trying to reveal the molecular mechanism underlying these 

observations. 

57. RELATIVE BIOLOGICAL EFFICIENCY OF PROTONS AT LOW AND THERAPEUTIC 

DOSES IN INDUCTION OF γH2AX FOCI AND APOPTOSIS 

Lucian Zastko 1,2 , S. Sorokina 2,3 , P. Plavckova 1,2 , E. Markova 2 , J. Gursky 2 , J. Dobrovodsky 4 , 

I. Y. Belyaev 2 

1 

Proton Therapy Complex, Central Military Hospital, Ružomberok, Slovak Republic; 

2 

Cancer Research Institute, Slovak Academy of Sciences, Bratislava, 

Slovak Republic; exonzast@savba.sk 

3 

Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, 

Pushchino, Russia 

4 

Slovak Institute of Metrology, Bratislava, Slovak Republic 

Currently, the radiation therapy of tumors is mostly performed with high-energy photon 

beams generated by clinical linear electron accelerators. High-energy proton or ion 

beams have recently been advanced as a promising alternative to photon radiotherapy 

treatment and is a rapidly emerging treatment modality. The unique properties of 

protons, which lose their energy forming a Bragg peak, enable precise delivery of a high 

dose of radiation to the tumor region, while also sparing critical organs and healthy 

tissues. However, this type of radiotherapy has not been widely accepted mainly because 

of high costs and related secondary neutron irradiation associated with increased risks 

of secondary cancers (Sorokina et al., 2013). 

New ProTom proton therapy technology, which may overcome some major obstacles 


for application of proton therapy, has recently become available at the Central Military 

Hospital Ružomberok, Slovak Republic. This technology uses pencil proton beams of 

different energies in the range of 30–300 MeV during the same treatment. Under ProTom, 

the combination of proton beams/energies is determined that covers the tumor by the 

correspondent Bragg peaks, while sparing normal tissue from damage. Such treatment 

will kill selectively cancer cells within the tumor while cells in normal tissues around 

the tumor are not significantly damaged. It is important to pinpoint the actual relative 

biological effectiveness (RBE) value that is defined relative to photon radiation (most 

often γ 60 Co-rays or linear accelerator X-rays). 

We measured RBE of 200 MeV protons in the Bragg peak relative to γ 60 Co-rays in 

human umbilical cord blood cells (UCBC). UCBC were obtained from healthy newborns. 

UCBC represent subsets of mononuclear cells: hematopoietic stem cells, precursor 

lymphocytes, mature lymphocytes, erythroblasts, and monocytes. 

Cellular FITC (histone γH2AX) was measured and analyzed with a BD FACSCanto II flow 

cytometer (BD Biosciences) and automatic fluorescent microscope Metafer Scanning 

System (Metasystems). Apoptosis was measured with Accuri C6 flow cytometer (BD 

Biosciences) and analyzed by quantifying the annexin V-FITC and propidium iodide (PI) 

signals. Statistically significant induction of γH2AX foci was seen at doses of 50 cGy and 

higher both for γ-rays and protons using flow cytometry (Fig. 1). Similar to data obtained 

with focus enumeration by Metafer, protons induced slightly higher effects at doses 

lower 20 cGy, but these effects were not statistically significant. While no statistically 

significant effect of radiation quality was seen by analysis of variance, increased RBE 

for protons at low doses < 20 cGy was consistently observed by all end-points. Level 

of early apoptotic cells 24 hours after γ-irradiation was similar at low (2, 20 cGy) and 

therapeutical (2 Gy) dose which is usually used in one fraction for treatment of various 

tumors. For late apoptosis, the data have shown almost 2-fold difference between low 

and therapeutical doses. Whether these data are comparable to proton-irradiation 

apoptotic effects, will be investigated later. 

In conclusion, our data provide evidence that γ-rays and protons induce similar number 

of ionizing radiation-induced foci in UCBC, as measured after therapeutic doses by 

fluorescence microscopy and flow cytometry. Further experiments will reveal if these 

findings match with apoptosis induction. 


This work was supported by the Structural Funds of EU (Agency) by the Ministry of 

Education, Science, Research and Sport of the Slovak Republic (Protonbeam, ITMS: 

26220220129), the Slovak Research and Development Agency (APVV 0669-10), the 

National Scholarship Program of the Slovak Republic (SAIA); and the VEGA Grant 

Agency (2/0150/11) of the Slovak Republic. 

References 

Sorokina, S., Markova, E., Gursky, J., Dobrovodsky, J., and Belyaev, I.: Relative biological 

efficiency of protons at low and therapeutic doses in induction of 53BP1/γH2AX foci 

in lymphocytes from umbilical cord blood, International Journal of Radiation Biology, 

2013 


Fig. 1. Radiation-induced γH2AX in UCBC irradiated with protons at doses of 5, 50 and 


200 cGy and fixed 30 min after irradiation for measurements using flow cytometry. 

Sectors Q3 represent control level of γH2AX, sectors Q4 contain cells with increased 

γH2AX intensity. γH2AX signal was measured as FITC intensity (X-values). Y-axis shows 

side scatter of cells. 

58. TUMOR-DRIVEN CHANGES IN HUMAN MESENCHYMAL STROMAL CELLS CAN 

GENERATE PHENOTYPE OF CARCINOMA-ASSOCIATED FIBROBLASTS 

Lucia Kucerova 1 , Jakub Zmajkovic 2 , Lenka Baranovicova 1 , Svetlana Skolekova 1 , Miroslava 

Matuskova 1 

1 

Laboratory of Molecular Oncology, Cancer Research Institute, Slovak Academy of 

Sciences, Vlarska 7, 833 91 Bratislava, Slovak Republic, lucia.kucerova@savba.sk 

2 

Arsanis Biosciences GmbH, Helmut-Qualtinger-Gasse 2, 1030 Vienna, Austria 

Background: Human mesenchymal stromal cells have been added to the list of numerous 

non-malignant cells contributing to tumor microenvironment thereby influencing tumor 

growth and progression. Tumor-produced paracrine factors are capable of driving 

molecular changes leading towards the differentiation of MSC into cells with the 

properties of carcinoma associated fibroblasts. MSC interaction with particular tumor 

cells is either tumor supportive or suppressive, and the underlying mechanisms remain 

to be elucidated [1-3]. We hypothesize that it is the tumor-dictated process of molecular 

changes in MSC responsible for the tumor growth support/inhibition. 

Methods: Project should unravel effects of tumor produced paracrine factors on 

MSC properties, such as differentiation capacity, immunophenotype and cytokine 

expression profile. More importantly, changes in production of proteins affecting tumor 

vascularization, aggressiveness, invasiveness and other sequence of events leading 

toward acquired carcinoma associated fibroblast phenotype in MSC dictated by tumor 

microenvironment were evaluated. These events were mimicked by long-term culture 

of MSC under the influence tumor-derived paracrine factors in vitro. Moreover, these 


effects will be also characterized upon MSC coimplantation with the tumor cells in vivo 

in the future. (Fig. 1). 

Figure 1. Experimental design and the outcomes to be evaluated in the process of 

tumor cell-driven MSC differentiation. 

Results: Our data previously documented both tumor-promoting and tumor-suppressive 

effects of MSC on different human tumor cells both in vitro and in vivo. Human melanoma 

cells A375 represented the promoting features and induced expression of VEGFR2, 

FSP, vimentin, αSMA and endosialin in MSC, which were indicative of differentiation 

towards carcinoma-associated fibroblasts. Human glioblastoma cells 8-MG-BA 

represented the inhibitory features and did not upregulate these proteins. On the 

contrary, paracrine stimulation with these cells led to upregulation of genes involved in 

adipocyte differentiation leading to the idea, that these cells promote aberrant terminal 

differentiation of MSC with the tumor-suppressive outcome. Several surface markers 

characteristic for the MSC were evaluated and multiple changes in CD73, CD90, CD105, 

CD133, CXCR4, CD166, CD146, BCRPI, HLA-DR, CD44, CD31 and CD34 III were detected. 

Tumor cells also affected migratory properties of MSC and altered the secretome profile 

of the treated MSC. Many proinflamatory cytokines were upregulated significantly by 

incubation in the A375-conditioned medium, including VEGF secretion and VEGFR2 

expression. 

Conclusion: Our data attempted to contribute to the understanding of tumor biology by 

unraveling mechanisms involved in tumor-driven effects on non-neoplastic stromal cells. 


Furthermore, they may unravel targets to interfere with in order to affect tumor stromal 

interaction and/or inhibit tumor growth. Moreover, counteracting inhibitory effects 

may enable to grow tumors and provide novel in vivo models as well as combination of 

differentiated tumor associated fibroblasts with tumor cells may produce more relevant 

tumor models for evaluation of tumor behavior and responses in vivo. 


This work was supported by the Slovak Research and Development Agency under the 

contract No.APVV-0230-11, VEGA grants 2/0088/11 and 2/0171/13. The experiments on 

the IncuCyte ZOOM were enabled with the kind help and the financial support from 

the Cancer Research Foundation. 

1. Kucerova L, Matuskova M, Hlubinova K, Altanerova V, Altaner C: Tumor cell behaviour 

modulation by mesenchymal stromal cells. Mol Cancer 2010, 9:129. 

2. Kucerova L, Kovacovicova M, Polak S, Bohac M, Fedeles J, Palencar D, Matuskova M: 

Interaction of human adipose tissue-derived mesenchymal stromal cells with breast 

cancer cells. Neoplasma 2011, 58:361-370. 

3. Kucerova L, Skolekova S: Tumor microenvironment and the role of mesenchymal 

stromal cells. Neoplasma 2013, 60:1-10. 

59. USE OF NANO-SIZED REALGAR PARTICLES AND SOLUBLE ARSENIC IN TREATMENT 

OF CANCER: KNOWLEDGE FROM TRADITIONAL CHINESE MEDICINE 

Pastorek M. 1 , Balaz P. 2 , Bujnakova Z. 2 , Jakubikova J. 1,3 , Cholujova D. 1 , Gronesova P. 1 , 

Duraj J. 1 , Hunakova L. 1 , Sedlak J. 1 

1 

Cancer Research Institute, Slovak Academy of Sciences 

2 

Institute of Geotechnics, Slovak Academy of Sciences 

3 

Dana Farber Cancer Institute, Department of Medical Oncology, Boston, USA 

Despite significant progress that has been made in field of cancer treatment, it still 

remains an incurable disease. The use of natural compounds from traditional chinese 

medicine and their application along with western knowledge is among a new emerging 

efforts in cancer patient treatment. Xionghuang is a traditional remedy containing realgar 

(As 4 

S 4 

) prepared by special procedure. Because of its very low solubility we have used 

high-energy milling to prepare realgar in form of nanoparticles. Therefore the effect of 

realgar was compared with ATO (arsenic trioxide) using the panel of melanoma cell lines. 

Flow cytometry analysis showed time- and concentration-dependent cytotoxicity 

of realgar nanoparticles and ATO solution that was proportional to concentration of 

arsenic. Both tested compounds influenced cell cycle distribution with significant 

induction of G2/M block. Despite postive and negative regulation of glutathione level 

observed within different ranges of arsenic concentration, the use of BSO (inhibitor of 

GSH synthesis) increased toxicity of realgar and ATO within whole concentration scale of 

arsenic. Combined treatment of realgar or ATO with sulforaphane, the chemopreventive 

compound known for modulation of GSH level, achieved synergistic toxic effect. 


Although multiple signalling pathways are known to be modulated by arsenic 

compounds, precise mechanism of action of realgar nanoparticles remains unclear. Even 

though physical properties of soluble ATO and realgar nanoparticles differ significantly, 

suprisingly substantial similarities in their effect on tested cells were observed. 


This study is the result of the project implementation: TRANSMED 2, ITMS 26240120030 

supported by the Research & Development Operational Programme funded by the ERDF 

and the Joint Research Projects Cooperation of SAS-NSC No. 2010/03 

60. EPIGENETIC EFFECTS OF VPA ON EXPRESSION OF CD133 IN NEUROBLASTOMA 

CELL LINES IN NORMOXIA AND HYPOXIA 

Ashraf M. Khalil, Jan Hraběta, Helena Maříková, Tomáš Groh, Šimon Cipro, Tomáš 

Eckschlager 

Department of Paediatric Haematology and Oncology, 2 nd Faculty of Medicine and 

University Hospital Motol, Charles University, Prague, Czech Republic 

CD133, also known as Prominin-1, is a pentaspan transmembrane glycoprotein 

commonly found in the stem and progenitor cells in various tissues particularly in the 

CNS. Many authors have shown the cancer stem cell (CSC) characters of CD133 positive 

cells in different solid tumours (Qianga et al., 2009). The value of studying such cells is 

related to the assumption they likely can initiate the tumour, relapse and metastasis. 

This is due to their ability for self-renewal, high proliferative potential, maintaining and 

regulating the neoplastic clone. Targeting CSCs by conventional therapy isn’t efficient to 

eradicate them because they are generally resistant to conventional chemotherapy and 

radiotherapy. 

Increased CD133 expression is described as a sign associated with worse prognosis 

in Neuroblastoma (NBL) and other tumours (Tong et al., 2008). NBL, a tumour of the 

peripheral sympathetic nervous system, is the most common extracranial solid tumour 

in children. Prognosis of high risk tumors is still poor, because drug resistance arises in 

the majority of those patients with a little improvement in therapeutic options during 

the last decade. Nowadays, epigenetic therapy is considered as a promising approach to 

target CSC. Histon deacetylase inhibitors (HDACi) are tested as a new class of anticancer 

agents which can induce chromatin remolding with subsequent re-expression of 

epigenetically silenced genes in tumours. Valproic acid (VPA) is an established drug in the 

long-term therapy of epilepsy. It has demonstrated antitumor activity as a (HDACi) such 

as inhibition of the cell cycle as well as induction of apoptosis. Epigenetic modifications 

such as Histon acetylation and promoter methylation have been proved to control CD133 

transcription (Tabu et al., 2008). Hence, we were interested in evaluating the effect of 

VPA on the expression of CD133 in normoxia and hypoxia 

In this study, we have examined 4 neuroblastoma cell lines. Neither IMR-32 nor UKF- 

NB4 were expressing CD133 protein whether in the control or sample treated with VPA 

as reveled by westernblot. Interestingly, CD133 transcription has been reactivated and 


CD133 protein was detected after using demethylating agent 5-aza-2’-deoxycytidine. 

Flowcytometric and western blot analysis showed that VPA treated culture increased 

expression of CD133 protein to several folds and maintained it high as far as VPA is 

included in the culture of SHSY5Y, UKF-NB3 NBL cell lines. On the other hand, Valpromid, 

a carboxamide derivative of VPA that lack the histone deacetylase inhibition effect 

doesn‘t induce CD133 expression in UKF-NB3 which confirms the regulation of CD133 

by the state of histone. 

We also highlight that hypoxia (1% oxygen) induced robust down regulation of CD133 

up to disappearance of the protein expression in UKF-NB3 cell line. Hypoxia-inducible 

factors seem to be involved in this regulation because the same effect was obtained 

when cobalt chloride, that mimic hypoxia by inducing hypoxia-inducible factors, was 

added to culture in normoxia. Moreover, the great effect of VPA on expansion of 

CD133 in normoxia, was diminished in hypoxic condition and is regained again when 

VPA is combined with 5-aza-2’-deoxycytidine, suggesting that part of the cells acquired 

methylation of the CD133 promoter in hypoxic condition. 

The regressive response of the amount of CD133 in hypoxia is unusual to CSCs behavior 

in comparison to CD133 positive cells in other solid tumours (Soeda et al., 2009) that 

is of great importance to understand the tumor microenvironment and the different 

reaction of CD133 expression in various cell types in response to hypoxia. Obviously, 

CD133 gene expression is directly controlled by epigenetic regulation. 

Acknowledgments 

This work was supported by GAUK grant No 620612 and GAČR No No P301/10/0356. 

Refrences 

Qianga L, Yanga Y, Ma YJ, et al.,: Isolation and characterization of cancer stem like cells in 

human glioblastoma cell lines. Cancer Letters 279: 13–21 (2009). 

Soeda A, Park M, Lee D, et al.: Hypoxia promotes expansion of the CD133-positive glioma 

stem cells through activation of HIF-1a. Oncogene 28: 3949–3959 (2009). 

Tabu K, Sasai K, Kimura T, et al.: Promoter hypomethylation regulates CD133 expression 

in human gliomas. Cell Research 18:1037-1046 (2008). 

Tong QS, Zheng LD, Tang ST, et al.: Expression and clinical significance of stem cell marker 

CD133 in human neuroblastoma. World J Pediatr 4(1):58-62 (2008) 

61. POPULATION DYNAMICS OF SP-PHENOTYPE UNDER IN VITRO CONDITIONS 

Jaromír Mikeš, Jana Vargová, Lucia Mikešová, Zuzana Zsihovicsová, Ján Kovaľ, Rastislav 

Jendželovský, Peter Fedoročko 

Institute of Biology and Ecology, University of Pavol Jozef Šafárik in Košice, Slovak Republic; 

jaromirmikes@yahoo.com 

A concept of cancer cells with “mighty” powers of stem cells was primarily identified and 

issued in haematological malignancies in 1990’s and it seems to be valid in solid tumour, 

too. The main consequence accrued from this concept is the existence of rare cells, 

aka “cancer stem-like cells” (cSCs), with a high resistance to anti-cancer therapy and 


an ability to recreate the tumour from one or limited number of surviving cells. These 

subpopulations, therefore, are hold responsible for relapses. Accordingly, it is presumed 

that therapy targeted against these cells should lead to better therapeutic outcome. 

Since these cells can be extremely rare, they are hard to identify or study. Moreover, 

there is a model of “hierarchical structure of tumours” that puzzles the whole situation as 

it proposes existence of multiple subpopulation with different phenotypes having different 

potential to prosper under particular conditions within the same tumour. There are multiple 

approaches to identify these cells based on molecular and/or physiological phenotype. 

One of these approaches is based on intensive efflux of specific substrates in cells 

with highly expressed/active ABC-transporters, which are often linked with multi-drug 

resistance (MDR) phenotype. Method based on efflux of DNA-binding fluorescent dye 

Hoechst 33342 allows to identify so called “side population” (SP) that can be found in 

multiple species (Goodell,1997). Although not all of these cells are exclusively presenting 

attributes of the stem cells or cSCs, this SP-population tends to be enriched with 

hematopoietic stem cells (Goodell, 1996) or these cells tend to be, at least, resistant to 

multiple drugs in case of tumour-originating cells (Hadnagy, 2006). As the method itself 

is rather empirically established, we focused the particular steps in order to more closely 

understand the basic principles. 

Comparing multiple cancer cell lines, we found significant variations in percentage of 

SP-cells ranging from less than 0.1 % to more than 50 %. Interestingly, we found as well, 

that the cultivation conditions, especially actual cell population density, are significantly 

affecting the final results. At the same time, we optimized the staining conditions and 

proved that thermal stability during staining significantly increases viability as well 

as ratio of SP-cells. However, we also found that variations in staining density of cells 

(100.000 – 5.000.000 cells/ml) results in enormous differences of SP-cells ratio (~2 – 80 

%). Changes in cultivation medium evoked by purposeful modifications or unintentionally 

by intensive usage of the incubator affected the overall outcome significantly as well. 

All these findings indicate that variable results presented in literature can be, at least 

partially, affected by any of the factors or their combination. 

Our results also demonstrates another important conclusion that the SP-phenotype in 

lung adenocarcinoma A549 cells cannot be “inherited” but it is rather given by multiple 

factors defined by various culturing conditions, yet primarily by actual cell density. This 

definitely distinguishes the cSCs from physiological subpopulations of stem cells. 


This work was supported by the Slovak Research and Development Agency under 

contract Nos. APVV-0040-10 and VVCE-0001-07 and the Scientific Grant Agency of the 

Ministry of Education of the Slovak Republic under contract No. VEGA 1/0626/11. 

References 

Goodell MA, et al.: Isolation and functional properties of murine hematopoietic stem 

cells that are replicating in vivo. - J Exp Med 183(4):1797-806, 1996. 

Goodell MA, et al.: Dye efflux studies suggest that hematopoietic stem cells expressing 

low or undetectable levels of CD34 antigen exist in multiple species. - Nat Med 3(12): 

1337-45, 1997. 

Hadnagy A, et al.: SP analysis may be used to identify cancer stem cell populations. - Exp 

Cell Res 312(19):3701-10, 2006. 


62. GREEN FLUORESCENCE PROTEIN (GFP) MICE IN HEMATOPOIETIC STEM CELL 

TRANSPLANTATION EXPERIMENTS: THE POSSIBLE LIMITATIONS IN CHIMERISM 

QUANTIFICATION 

Filipp Savvulidi, Katarina Forgacova, Emanuel Necas, Ludek Sefc 

Charles University in Prague, 1 st Faculty of Medicine, Prague, Czech Republic, 

immunophi@gmail.com 

Transgenic mice expressing enhanced Green Fluorescent Protein (GFP) under the 

direction of the human ubiqutin C promoter (C57BL/6-Tg(UBC-GFP)30Scha/J mice) are 

frequently used in hematopoietic stem cell (HSC) transplantation experiments. GFP is 

expressed in the leukocytes, platelets, and erythrocytes of these mice. Bone marrow 

cells are transplanted into congenic C57BL/6 recipients and chimerism of white blood 

cells based on GFP expression is determined in lysed peripheral blood of recipient at 

different intervals after transplantation. Another congenic mouse transplantation model 

uses functionally identical isoforms of CD45 antigen - Ly5.1 (CD45.1) or Ly5.2 (CD45.2). 

GFP and their congenic C57BL/6 recipients both express just Ly5.2 isoform. 

In order to compare the accuracy of both models, as well as to establish a new model 

for tracking of mutual competition of three hematopoietic tissues together, we cotransplanted 

GFP(Ly5.2) and Ly5.1 bone marrow cells to sublethally irradiated wild-type 

C57BL/6 (Ly5.2) recipients. We expected to distinguish GFP + Ly5.1 - Ly5.2 + , GFP - Ly5.1 - 

Ly5.2 + and GFP - Ly5.1 + Ly5.2 - populations representing all three mouse strains involved. 

Surprisingly, we found also GFP + Ly5.1 + Ly5.2 - cells (5-10%) which were not doublets nor 

cells of any used strain. These cells were detected in lower number also in bone marrow 

of transplanted mice which was not lysed in contrary to peripheral blood. 

In order to explain the presence of evidently false, Ly5.1/GFP double-positive cells in 

our observation, we incubated non-GFP Ly5.1 peripheral blood cells with supernatant 

from lysed GFP blood. A significant portion of Ly5.1 cells gained GFP positivity (Fig. 1B). 

Filtering of GFP supernatant with 0.22 µm filter abolished its GFP staining capacity (Fig. 1C). 

We conclude that fragments of GFP cells after ammonium chloride lysis, presumably of 

erythrocyte ghosts, attach at the surface of Ly5.1 cells generating artificial Ly5.1/GFP 

double-positive particles. This cellular debris is relatively large in size (larger than 0.22µm). 

Our results demonstrate that using a standard GFP - C57BL/6 congenic transplantation 

model, a GFP derived chimerism can be overestimated by flow cytometry detection. 


We thank M. Molik for his excellent technical help. 

This work was supported by the Ministry of Education, Youth and Sports of the Czech 

Republic (project PRVOUK-P24/LF1/3), and by Charles University in Prague (grant SVV- 

2012-264507) 

Legend to Fig.1: Peripheral 

blood cells, singlets gated. (A) 

Ly5.1 cells stained with anti- 

Ly5.1 Ab; (B) Ly5.1 cells co- 


incubated with non-filtered supernatant of lysed GFP cells; (C) Ly5.1 cells co-incubated 

with 0.22um filtered supernatant of lysed GFP cells; (D) unstained GFP cells. 

63. NORMAL HEMATOPOIETIC STEM CELLS CAN BE DETECTED IN NEWLY DIAGNOSED 

CHRONIC MYELOID LEUKEMIA PATIENTS AND LEUKEMIC STEM CELLS CAN BE 

DETECTED IN CML PATIENTS AFTER THERAPY BY A MULTI-TECHNIQUE APPROACH 

USING ANTIGEN CD26 

Marek Borský 1 , Veronika Neméthová 1 , Filip Rázga 1 , Jiří Mayer, Zdeněk Ráčil 1,2 

1 

University Hospital of Brno, mborsky@fnbrno.cz 

2 

Masaryk University Brno 

Despite the success in the treatment of CML with tyrosine kinase inhibitors, these drugs 

cannot eradicate the disease because the most primitive, quiescent leukemic stem cells 

(LSC) survive. The LSCs as well as their offsprings are characterized by the presence of 

the fusion oncogene BCR-ABL. BCR-ABL is a specific cytogenetic abnormality known as 

the Philadelphia chromosome (Ph). Ph + cells constitute the majority of leukocytes in 

bone marrow (BM) in CML patients at diagnosis. How can we distinguish LSC and normal 

hematopoietic stem cells (HSC)? Several studies have shown differences in expression 

of some plasma membrane-associated genes between CML LSC and normal HSC. These 

include cell surface genes significantly upregulated in CML LCSs: CD26, CD25, PTPRD, 

CACNA1D, IL1RAP and other (Gerber et al. 2013). Investigation of CD26 disclosed that 

the engraftment of Lin - /CD26 + stem cells into NOD scid gamma mice resulted in BCR/ 

ABL + cells expansion whereas the engraftment of Lin - /CD26 - stem cells lead to normal 

multilineage BCR/ABL - hematopoiesis. 

We aimed to identify CML LSC and HSC using anti-CD26 in BM of CML patients at diagnosis. 

For this purpose we established protocol combining MACS, FACS and FISH techniques. 

In the first step, neutrophils were depleted by anti-CD15 mAbs using MACS which lead 

to about ten times higher concentration of CD34 + cells in the sample. Subsequent cell 

sorting separated target subpopulations CD45 + 34 + 38 - , CD45 + 34 + 38 + , CD45 + 34 + 38 - 26 - and 

CD45 + 34 + 38 - 26 + for the purpose of Ph-positivity evaluation using FISH technique. 

Our preliminary data from 15 newly diagnosed chronic myeloid leukemia patients suggest 

the existence of two expression patterns. In pattern 1, antigen CD26 is a discriminatory 

marker for distinguishing Ph + cells from Ph - cells. This pattern is characterized by presence 

of at least 20% or more small cells (FSC/SSC low ) which are predominantly CD45 + 34 + 38 - 

26 - and, at the same time, Ph - (non-malignant HSC). In pattern 2, the antigen CD26 is 

expressed on both Ph + and Ph - cells and the proportion of FSC/SSC low cells is lower than 

20%. Our findings are similar to results of Holland’s research group (Janssen et al.2012). 

In this work, patients with pattern 2 appear to have higher risk due to higher Euro score. 

Following these observation we started separation of target subpopulations from BM 

of monitored patients after therapy. We aimed to find differences between patients 

with pattern 1 compared to patients with pattern 2 which develop after therapy. Our 

preliminary results show that CD45 + 34 + 38 - Ph - cells are exclusively CD26 - , whereas Ph + 


cells (LSC) are present in CD45+34+38-26+ subpopulation only. Loss of expression of 

CD26 on Ph - cells from patients with pattern 2 appears to be specific for after-therapy 

phase of the disease. On the other hand, not all CD45 + 34 + 38 - 26 + cells were found Ph 

positive. 

Our findings confirm that CD26 antigen is useful for discrimination between Ph + and 

Ph - cells in exact disease phase and/or pattern-dependent only. However, considering 

easy identification of chronic myeloid stem cells by Ph positivity in CD45 + 34 + 38 - 26 - or 

CD45 + 34 + 38 - 26 + populations, it is obvious that precise separation LSC from HSC in target 

populations using CD26 is not possible in all settings. Despite these facts, this approach 

opens up a new possibilities for CML study. 


Supported by MH CZ - DRO (FNBr, 65269705) 

References 

Gerber JM, Gucwa JL, Esopi D, Gurel M, Haffner MC, Vala M, Nelson WG, Jones RJ, 

Yegnasubramanian S.: Genome-wide comparison of the transcriptomes of highly 

enriched normal and chronic myeloid leukemia stem and progenitor cell populations. 

Oncotarget. 2013 

Janssen JJ, Deenik W, Smolders KG, van Kuijk BJ, Pouwels W, Kelder A, Cornelissen JJ, 

Schuurhuis GJ, Ossenkoppele GJ.: Residual normal stem cells can be detected in newly 

diagnosed chronic myeloid leukemia patients by a new flow cytometric approach and 

predict for optimal response to imatinib. Leukemia 2012 

64. EPITHELIAL-TO-MESENCHYMAL TRANSITION IN SUBPOPULATION OF MOUSE 

PROSTATE STEM CELLS AND CANCER STEM-LIKE CELLS 

Zuzana Pernicová 1,2 , Eva Slabáková 1,2 , Radek Fedr 1 , Maximilian Marhold 3 , Erwin 

Tomasich 3 , Peter Horak 3 , Alois Kozubík 1,4 , Karel Souček 1,2 

1 


Republic, Brno, Czech Republic; E-mail: pernicova@ibp.cz 

2 

Center of Biomolecular and Cellular Engineering, International Clinical Research Center, 

St. Anne‘s University Hospital Brno, Brno, Czech Republic 

3 

Department of Internal Medicine I – Oncology, Comprehensive Cancer Center, Medical 

University of Vienna, Wien, Austria 

4 

Department of Experimental Biology, Faculty of Sciences, Masaryk University, Brno, 


Epithelial-to-mesenchymal transition (EMT) is an important process during 

embryogenesis, which may be re-activated in several pathophysiological processes 

including tumor progression. In several models, normal cells or cancer cells undergoing 

EMT display properties and characteristics of stem cells (Kong et al., 2011; Mani et al., 

2008). In our work, we aimed to identify EMT transcription factors with altered expression 

in subpopulation of mouse prostate stem cells or cancer stem cells, respectively, to prove 

involvement of EMT in biology of stem cells also in mouse prostate. There are several 


approaches how to detect and isolate mouse prostate stem cells and cancer stem cells, 

respectively. According to published results (Goldstein et al., 2008), using multicolor 

flow cytometry we detected putative mouse prostate adult stem cells with phenotype 

Lin - /Sca1 + /CD49f + /Trop-2 + in normal prostate and prostate cancer. 

To elucidate whether subpopulation of cells expressing stem cells markers display 

changes in levels of EMT markers and regulators, using high speed sorter we isolated 

subpopulations of cells with phenotype Lin - /Sca1 + /CD49f + /Trop-2 + and Lin - /Sca-1 - /CD49f - . 

Further we introduced a PCR method for detection of mRNA level from limited number 

of sorted cells and compared mRNA levels of EMT regulators (SNAI1, SNAI2, TWIST2, 

ZEB1, ZEB2), and markers of differentiation (VIM and CDH1) in these subpopulations. 

Our results show that EMT transcription factor SNAI2/Slug is strongly up-regulated in 

subpopulation of both mouse prostate stem cells and cancer stem cells with phenotype 

Lin - /Sca1 + /CD49f + /Trop2 + . Interestingly, this was not accompanied with up-regulation of 

EMT markers, since vimentin was found to be strongly down-regulated in both mouse 

prostate stem cells and cancer stem cells subpopulations. Further we aimed to confirm 

this Slug up-regulation also on protein level using multicolor flow cytometry protocol for 

simultaneous detection of selected surface and intracellular markers. 

Taken together our results show that subpopulation of cells with stem cell characteristics 

can be detected in both wt prostate and prostate cancer using combination of surface 

markers Sca-1, CD49f and Trop-2. Moreover, we found out that transcription factor and 

regulator of EMT Slug is strongly up-regulated in this subpopulation of putative prostate 

stem cells / cancer stem cells. 


This work was supported by grants IGA MZD NT13573-4/2012, AV ČR M200041203, GA 

ČR P301/12/P407, HistoPARK (CZ.1.07/2.3.00/20.0185), and by project FNUSA-ICRC (no. 

CZ.1.05/1.1.00/02.0123) from the European Regional Development Fund. Institutional 

support was provided by the Academy of Sciences of the Czech Republic. 

References 

Goldstein, A. S., Lawson, D. A., Cheng, D., Sun, W., Garraway, I. P., and Witte, O. N.Trop2 

identifies a subpopulation of murine and human prostate basal cells with stem cell 

characteristics: Proc Natl Acad Sci U S A, v. 105, p. 20882-7, 2008. 

Kong, D., Li, Y., Wang, Z., and Sarkar, F. H. Cancer Stem Cells and Epithelial-to-Mesenchymal 

Transition (EMT)-Phenotypic Cells: Are They Cousins or Twins?: Cancers (Basel), v. 3, p. 

716-729, 2011. 

Mani, S. A., Guo, W., Liao, M. J., Eaton, E. N., Ayyanan, A., Zhou, A. Y., Brooks, M., 

Reinhard, F., Zhang, C. C., Shipitsin, M., Campbell, L. L., Polyak, K., Brisken, C., Yang, J., and 

Weinberg, R. A. The epithelial-mesenchymal transition generates cells with properties of 

stem cells: Cell, v. 133, p. 704-15, 2008. 


66. DNA REPAIR STUDIES IN LIVING CELLS BY THE USE OF CONFOCAL MICROSCOPY 

Eva Bártová, Soňa Legartová, Petra Sehnalová, Lenka Stixová, Stanislav Kozubek 

Institute of Biophysics, Academy of Science of Czech Republic, v.v.i., Brno, Czech Republic; 

bartova@ibp.cz 

The maintenance of genome integrity is fundamental for proper cellular functions. 

Genomes are continuously exposed to genotoxic injuries, including UV irradiation 

and oxidative stress caused by pollutants. Thus, starting an appropriate DNA repair 

signaling pathway is more than demanding for genome stability. Genotoxic stress 

generally leads to induction of strand breaks in DNA and especially double-stranded 

breaks are dangerous in terms of their faulty repair. The result of inappropriate DNA 

repair is the emergence of mutations or chromosomal translocations leading to cancer 

progression. Thus, here, we tried to address the function of epigenetic factors, including 

histone acetylation, phosphorylation and methylation during DNA damage response. In 

addition, in living cells, we analyzed histone code-related proteins and their functional 

properties in relationship to optimal DNA repair. In tumor cells, we found acetylationdependent 

recruitment of BMI1 and HP1β proteins into locally induced DNA lesions. 

Moreover, appearance of Polycomb group-related BMI1 protein and heterochromatin 

protein HP1β to DNA lesions was ATPdependent. Changes in histone acetylation and 

phosphorylation of H2AX, we also studied in embryonic stem cells, characterized by 

acetylation-dependent recruitment of OCT4 protein to DNA lesions, induced in living 

cells by local micro-irradiation. Besides OCT4, transcription factors UBFs were recruited 

to DNA lesions induced in both nucleoli and non-nucleolar compartment of interphase 

nuclei. These experiments showed us a new direction of how to study the fate of 

transcription factors after micro-irradiation of living cells. 


Work was supported by Grant Agency of the Czech Republic, project No.: 13-07822S 

and by national COST-CZ project No.: LD11020. 

67. INTRODUCTION TO AUTOMATED MICROSCOPY FOR HIGH-THROUGHPUT AND 

HIGH-CONTENT SCREENING 

Eva Vesela 1 , Tomas Furst 2 , Lenka Radova 3 and Martin Mistrik 1 

1 

Laboratory of Integrity of Genome, Institute of Molecular and Translational Medicine, 

Palacky University, Olomouc, Czech Republic; eva.vesela@upol.cz 

2 

Department of Matematical Analysis and Math Apllications, Faculty of Science, Palacky 

University,Olomouc, Czech Republic 

3 

Laboratory of Experimental Medicine, Institute of Molecular and Translational 

Medicine, Palacky University, Olomouc, Czech Republic 

Microscopy-based readout provides the most detailed single cell evaluation. Recent 


progress in development of automated microscopes and specialized image analysis 

software opens new possibilities for application of microscopes in high-content (HCS) 

and high-throughput screenings (HTS). In this presentation an overview of current 

technologies will be introduced including practical experience using three different 

automated microscopes currently available in our institute. Automated high-content 

measurements are frequently used for evaluations of a wide range of parameters related 

to whole cells and/or its parts. Selected commercial and open-source software for image 

acquisition and analysis will be briefly introduced. Last but not least, several practical 

problems guiding sample preparation and data handling will be discussed. 

Acknowledgement: 

This work was supported by grant provided by Ministry of the Interior of Czech Republic 

No.VG2010201400 

70. DOWNREGULATION OF WIP1 PHOSPHATASE MODULATES THE CELLULAR 

THRESHOLD OF DNA DAMAGE SIGNALING IN MITOSIS 

Jan Benada 1,2 , Libor Macurek 1,2 , Erik Müllers 3 , Vincentius A. Halim 4 , Kateřina Krejčíková 2 , 

Kamila Burdová 2 , Soňa Pecháčková 1,2 , Zdeněk Hodný 2 , Arne Lindqvist 3 , 

René H. Medema 4 and Jiri Bartek 2,5 

1 

Department of Cancer Cell Biology and 2 Department of Genome Integrity, Institute of 

Molecular Genetics, Academy of Sciences of the Czech Republic, CZ14200 Prague, Czech 

Republic; jan.benada@img.cas.cz 

3 

Department of Cell and Molecular Biology, Karolinska Institutet, SE-17177 Stockholm, 

Sweden; 

4 

Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, 

Netherlands; 

5 

Danish Cancer Society Research Center, Copenhagen, Denmark 

Cells are constantly challenged by DNA damage and protect their genome integrity 

by activation of an evolutionary conserved DNA damage response pathway (DDR). A 

central core of DDR is composed of a spatiotemporally ordered net of posttranslational 

modifications among which protein phosphorylation plays a major role. Activation of 

checkpoint kinases ATM/ATR and Chk1/2 leads to stabilization of tumor suppressor p53, 

which results in a temporal arrest of cell cycle progression (checkpoint) and allows time 

for DNA repair (Bartek and Lukas, 2007). 

Several key proteins of DDR machinery localize directly to the site of DNA breaks and 

form distinct nuclear foci (Lukas et al., 2011). Their number reflects the level of DDR 

activation in a quantitative manner. The most commonly used markers of these foci 

are phosphorylated histone H2AX (γH2AX) and repair mediator protein 53BP1, which 

is recruited as a result of phosphorylation- and ubiquitination-dependent events. To be 

able to precisely determine the DDR foci number per cell, we applied the high-content 

image analysis approach using automated imaging station Scan^R (Olympus). 

Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 


phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR 

and is essential for timely termination of the DDR (Medema and Macurek, 2012). Here 

we have investigated how Wip1 is regulated in the context of the cell cycle (Macurek 

et al., 2013). Wip1 protein level increases from G1 phase to G2 and declines in mitosis. 

Decreased level of Wip1 during mitosis is caused by proteasomal degradation in APC/ 

C-cdc20-dependent manner. In addition, Wip1 is phosphorylated at multiple residues 

during mitosis and this leads to inhibition of its enzymatic activity. Importantly, ectopic 

expression of Wip1 reduced γH2AX foci number in mitotic cells and decreased the 

number of 53BP1 nuclear bodies in G1 cells. We propose that the combined degradation 

and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation 

and enables cells to react even to modest levels of DNA damage encountered during 

unperturbed mitotic progression. 

Acknowledgements: 

We are thankful to Dr. Staněk (IMG, Prague) for making the Scan^R imaging station 

available and all members of our laboratories for helpful discussions. This work was 

supported by the Grant Agency of the Czech Republic (projects P305/10/P420 and 

P301/10/1525), the Netherlands Genomic Initiative of NWO (CGC), the Danish Cancer 

Society and the European Commission (project DDResponse). AL was supported by 

Swedish Cancer Foundation and Swedish Childhood Cancer Foundation. 

References: 

Bartek, J. & Lukas, J. (2007), ‚DNA damage checkpoints: from initiation to recovery or 

adaptation.‘, Curr. Opin. Cell Biol. 19(2), 238-245. 

Lukas, J.; Lukas, C. & Bartek, J. (2011), ‚More than just a focus: The chromatin response 

to DNA damage and its role in genome integrity maintenance.‘, Nat Cell Biol 13(10), 

1161-1169. 

Macurek L, Benada J, Müllers E, Halim VA, Krejčíková K, Burdová K, Soňa Pecháčková, 

Zdeněk Hodný, Arne Lindqvist, René H Medema and Jiri Bartek (2013), ‚Downregulation 

of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in 

mitosis.‘ Cell Cycle; 12(2): 251–262. 

Medema, R. H. and Macurek, L. (2012), ‚Checkpoint control and cancer.’ Oncogene 

31(21), 2601-2613. 

71. ANALYSIS OF MAST CELL MOTILITY, MORPHOLOGY AND ACTIVATION USING 

QUANTITATIVE IMAGE-BASED CYTOMETRY 

Monika Bambousková, Ivana Hálová, Petr Dráber 

Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, 

Czech Republic; monika.bambouskova@img.cas.cz 

Mast cells are important component of innate and adaptive host immune system. They 

contribute to protection against helminths and bacterial infection and play a role in 

angiogenesis and autoimmunity. Under pathological conditions mast cells are involved in 

allergic and inflammatory diseases (1). Enhanced accumulation of mast cells in inflamed 


tissues is characteristic for a number of these pathological states. Thus, chemoattractantdirected 

migration of mature mast cells or their progenitors might be one of the key 

mechanisms responsible for local accumulation of these cells under physiological or 

pathological conditions. 

Aggregation of high-affinity receptors for IgE (FcεRI), expressed in the plasma membrane 

of mast cells, with polyvalent antigen (Ag) leads to cell activation and release of a 

broad spectrum of chemical mediators. FcεRI is also involved in mast cell chemotaxis 

towards Ag. Mast cells express a variety of chemokine receptors which bind various 

chemoattractants, including stem cell factor, sphingosin-1-phosphate, and arachidonic 

acid metabolites as leukotriens and prostaglandins (2). Prostaglandin E 2 

(PGE 2 

) is one 

of the major eicosanoids generated during inflammation and potent mediator which 

substantially influence mast cell responses, including chemotaxis (3). PGE 2 

binds to 

E-prostanoid receptors and engage cAMP/PKA pathway (4). However, detail signaling 

process leading to mast cell chemotaxis mediated by PGE 2 

is incompletely understood. 

In this study we analyzed the effect of different drugs affecting cAMP/PKA signaling 

in PGE 2 

stimulated mouse bone marrow derived-mast cells (BMMCs). We focused on 

characterisation of cell spreading on fibronectin as well as changes in cell motility after 

stimulation. For analysis of these responses we introduced several quantitative imagebased 

cytometry techniques which facilitate large scale image acquisition and data 

processing. Using these and other methods we found that potentiation or blocking of 

cAMP/PKA signaling has signifcant effect on mast cell signaling pathways induced by 

PGE 2 

. Consequently, these signaling pathways might contribute to pathological states 

occuring in allergy and inflammatory diseases. 


This work was supported by COST CZ project No. LD12073. 

References 

Galli, S. J., Tsai, M., and Piliponsky, A. M.: The development of allergic inflammation. – 

Nature 454: 445-454, 2008. 

Hálová, I., Dráberová, L., and Dráber, P.: Mast cell chemotaxis – chemoattractants and 

signaling pathways. - Frontiers in Immunology 3: 1-19, 2012. 

Kuehn, H. S., Rådinger, M., Brown, J. M., Ali, K., Vanhaesebroeck, B., Beaven, M. 

A., Metcalfe, D. D., and Gilfillan, A. M.: Btk-dependent Rac activation and actin 

rearrangement following FcεRI aggregation promotes enhanced chemotactic responses 

of mast cells. – Journal of Cell Science 123: 2576-2585, 2010. 

Serra-Pages, M., Olivera, A., Torres, R., Picado, C., de Mora, F., and Rivera, J.: E-prostanoid 

2 receptors dampen mast cell degranulation via cAMP/PKA-mediated suppression of 

IgE-dependent signaling. - Journal of Leukocyte Biology 92: 1155-1165. 2010. 


Fig. 1. Images and quantification of changes in mast cell morphology after activation 

with Ag or PGE 2 

. BMMCs were sensitized with IgE and attached to fibronectin. Cells 

were activated with Ag (B), PGE 2 

(C) or were not activated (A). After fixation and 

permeabilization the cells were stained with Alexa Fluor 488-phalloidin for filamentous 

actin (F-Actin; green) and with Hoechst stain for nuclei (blue). Images were acquired 

on Olympus Scan^R system and analyzed using CellProfiler image analysis software 


(Broad Institute, Boston MA). Ag-activated cells spread 

on fibronectin whereas upon PGE 2 

stimulation the 

cells created F-Actin rich protrusions. Cell area was 

determined and normalized to non-activated cells (D). 

At least 300 cells were evaluated in each condition. 

Means ±SD were calculated from duplicates in one 

representative experiment. Bars 20 mm. 

ABSTRACTS – POSTERS 

P1. NITRO-OLEIC ACID ATTENUATES THE INNATE IMMUNE RESPONSES OF 

MACROPHAGES STIMULATED WITH BACTERIAL ENDOTOXIN 

Gabriela Ambrozova 1,2 , Lukas Kubala 1,2 , Tanja K. Rudolph 3 , Antonin Lojek 1 , Thorben 

Ravekes 3 , Michaela Pekarova 1 

1 

Institute of Biophysics AS CR, Brno, Czech Republic; ambrozova@ibp.cz 

2 

ICRC/FNUSA, Brno, Czech Republic; 

3 

Hamburg University Heart Center, Hamburg, Germany 

Nitrated fatty acids (NO 2 

-FAs) represent a novel group of pluripotent signalling molecules, 

endogenously generated as an adaptive response of organism to oxidative stress (Baker 

et al., 2009). NO 2 

-FAs were shown to exert significant anti-inflammatory signalling action 

in immune and vascular models and so are hypothesized as protective compounds that 

can effectively down regulate ineligible activation of innate immune cells, particularly 

professional phagocytes (Trostchansky and Rubbo, 2008). Protective effects of NO 2 

-FAs 

on severe diseases including pulmonary hypertension and atherosclerosis have been 

described (Rudolph et al., 2010). Nevertheless, detailed mechanisms of their action in 

regulation of innate immune responses are not completely understood. Therefore, we 

investigated the role and mechanism of action of nitro-oleic acid (OANO 2 

) in endotoxinstimulated 

RAW 264.7 macrophages. 

Our results showed that OANO 2 

prevented the endotoxin-induced activation of 

macrophages in a dose-dependent manner. It was able to down-regulate phagocytic 

capability, production of reactive oxygen and nitrogen species as well as inflammatory 

cytokines. Importantly, changes in the production of inflammatory mediators were 

associated with reduction of endotoxin-induced MAPK activation and expression of NF- 


kappaB. Beside that, we found OANO 2 

-dependent inhibition of NADPH oxidase, iNOS, 

CD36, and TLR2/4 expression in endotoxin-stimulated RAW 264.7 cells. 

We can conclude that OANO 2 

is able to significantly reduce the oxidative and nitrative 

stress, generated within the macrophage-dependent innate immune responses, via the 

regulation of crucial intracellular signalling pathways connected with MAPK and NFkappaB 

activation. 

Acknowledgements: This work was supported by the grant of GAČR 13-40824P. 

References: 

Baker, P. R., Schopfer, F. J., O’Donnell, V. B., and Freeman, B. A.: Convergence of nitric 

oxide and lipid signaling: anti-inflammatory nitro-fatty acids, Free Radic Biol Med 46, 

989-1003, 2009. 

Rudolph, T. K., Rudolph, V., Edreira, M. M., Cole, M. P., Bonacci, G., Schopfer, F. J., 

Woodcock, S. R., Franek, A., Pekarova, M., Khoo, N. K., Hasty, A. H., Baldus, S., and 

Freeman, B. A.: Nitro-fatty acids reduce atherosclerosis in apolipoprotein E-deficient 

mice, Arterioscler Thromb Vasc Biol 30, 938-945, 2010. 

Trostchansky, A., and Rubbo, H.: Nitrated fatty acids: mechanisms of formation, chemical 

characterization, and biological properties, Free Radic Biol Med 44, 1887-1896, 2008. 

P2. THE ROLE OF AUTOPHAGY IN MODULATION OF 5-FU-INDUCED DEATH RESPONSE 

IN COLON CANCER CELLS WITH DIFFERENT P53 STATUS 

Barbora Bujokova 1,2 , Iva Jelinkova 1,2 , Birce Akpinar 3 , Alois Kozubik 1,2 , Boris Zhivotovsky 3 , 

Magnus Olsson 3 , Alena Hyrslova Vaculova 1 

1 


Republic, v.v.i., Brno, Czech Republic 

2 

Department of Animal Physiology and Immunology, Faculty of Science, Masaryk 

University, Brno, Czech Republic 

3 

Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, 

Stockholm, Sweden 

Autophagy is a cellular catabolic pathway by which macromolecules and organelles 

are sequestered in autophagosomes and delivered to the lysosomes for degradation. 

Autophagy may serve both, to promote cell survival or execute cellular demise. Autophagy 

plays an important role in carcinogenesis, and can also be induced by chemotherapeutic 

drugs in various tumor types. The role of autophagy in cancer therapy still needs to 

be clarified, as it has been shown either counteract or mediate the cytotoxic effects of 

some anticancer agents. Thus, selective modulation of autophagy may be considered as 

a novel and effective approach for enhancing the efficacy of existing anticancer therapy. 

5-Fluorouracil (5-FU) is a well-known chemotherapeutic drug used in the treatment of 

patients with colorectal cancer. Intracellular metabolites of 5-FU can exert their cytotoxic 

effects via inhibition of thymidylate synthase, or through incorporation into DNA and 

RNA, events that finally induce cell cycle arrest or apoptosis. In addition, autophagy 

has also been reported as an important mechanism in the cellular response to 5-FU. 


However, a precise mode of the regulation of 5-FU-elicited autophagy and its functional 

role in general cytotoxic response of colon cancer cells as well as its relationship with 

p53 signaling still remains poorly defined. 

We investigated the ability of 5-FU to trigger autophagy in human colon cancer cells with 

different p53 status, the kinetics of the response, and the essential molecules involved. 

Using chemical inhibitors of autophagy (e.g. 3-methyladenine, bafilomycin A1) or specific 

siRNAs against crucial autophagy regulators of the Atg family, we studied the functional 

role of autophagy in modulation of the overall death response of colon cancer cells to 

5-FU. The crosstalk of 5-FU-induced autophagy and apoptosis was also examined and 

compared in cells with different status of p53. 

We showed that the ability of 5-FU to trigger autophagic response was significantly 

enhanced in colon cancer cells deficient for p53, which were simultaneously less 

sensitive to 5-FU-induced apoptosis compared to their wt counterparts. Chemical 

inhibitor-mediated blockage of autophagy in its early (3-MA) or later (bafilomycin 

A1) stages resulted in suppression or stimulation of 5-FU-induced apoptosis and/or 

overall death especially in p53-deficient colon cancer cells, respectively. At the same 

time, siRNA-mediated silencing of selected Atg family regulators (e.g. Beclin 1, Atg7) 

exerted weaker effects. In addition, inhibition of selected MAPKs impaired 5-FU-induced 

autophagy preferentially in cells lacking p53. 

Our results suggest that targetting autophagy could contribute to the modulation of the 

general cytotoxic response to 5-FU especially in colon cancer cells with non-functional 

p53. 

This work was supported by the grants from IGA of the Ministry of Health of the Czech 

republic No. NT11201-5, Ministry of Education, Youth and Sports of the Czech Republic, 

European Regional Development Fund (CZ.1.07/2.3.00/20.0180). 

P3. EFFECT OF HSP90 INHIBITION ON CELL CYCLE REGULATION VIA ITS CLIENT 

PROTEINS 

Ľubomír Čulka, Jaromír Mikeš, Lenka Kundeková, Peter Fedoročko 

P. J. Šafárik University in Košice, Faculty of Science, Institute of Biology and Ecology, 

Košice, Slovak Republic; lubo.culka@gmail.com 

Heat shock protein 90 (HSP90) is an abundant protein crucial for many cellular processes. 

It fulfils a protective function for the cell under stress conditions and it also plays an 

essential role as molecular chaperone. In addition, HSP90 exerts its function via specific 

subset of proteins called client proteins, as the HSP90 function is essential for their 

conformational maturation and stability. Regulation of client proteins by the chaperone 

plays a crucial role in processes such as cell cycle control, differentiation and apoptosis 

(Mahalingam et al., 2009) 

In cancer cells, HSP90 overexpression is regularly observed in many types of tumours 

and its higher levels are typically associated with poorer prognosis and resistance to 


therapy in patients (Calderwood et al., 2006). Altered function of HSP90 as well as its 

higher levels seem to be essential for survival of cancer cells. Deregulation of HSP90 

and destabilization of its client proteins, which are known to be components of complex 

signalling pathways, contribute to „hallmarks of cancer“ (Bagatell and Whitesell, 2004). 

HSP90 is involved in regulation of cell cycle transition which, among others, is mediated 

by client proteins- Aurora B and CHK1 kinase. Both client proteins are characteristic 

for G2/M and M phase of cell cycle and they are often found to be overexpressed in 

colon cancers. As cancer cells require higher levels of HSP90, the chaperone has become 

attractive target for anticancer drug and several strategies to suppression of HSP90 level 

have been documented, including specific inhibitors of HSP90 such as geldanamycin 

or its derivatives (17-AAG, 17-DMAG), the agents targeting the conserved ATP-binding 

domain in molecular structure of the chaperone, leading to disruption of HSP90 and its 

client proteins (Li et al., 2012). 

Photodynamic therapy (PDT) is an alternative approach used as anti-cancer treatment 

with selective toxicity towards tumour cells utilizing oxygen, photosensitizer and light 

of specific wave length. Singlet oxygen and reactive oxygen species (ROS) produced 

during photodynamic reaction evoke destruction on molecular level targeting, among 

others, also the HSP90 protein (Dougherty et al., 1998). Hypericin is naturally occurring 

photosensitizer found in Hypericum perforatum plant and is used in PDT. 

Involvement of HSP90 and its client proteins (Aurora B, CHK1) in regulation of cell cycle 

in cancer cells in context of photodynamic therapy with hypericin or 17-DMAG is the aim 

of our study. 

Initially, we performed an extensive screening using a panel of tumour cell lines that 

were subjected to hypericin-mediated PDT (HY-PDT) with various doses of hypericin and 

time intervals. The treatment revealed alterations in distribution of cells in each phase 

and also cell cycle arrests in several cell lines. Based on significant and relevant changes 

in cell cycle, we selected the cell lines arrested in G2/M phase (colon and colorectal cell 

lines) in order to examine changes in levels of HSP90 protein and its client proteins. 

Surprisingly, analyses of three cancer cell lines (HCT116, HT29, SW620) exposed to HY- 

PDT or 17-DMAG did not show significant alterations in levels of HSP90. Similarly, levels 

of Aurora B seemed to be moderately influenced. But the treatments markedly lowered 

the CHK1 level. 

Further analyses revealed significant decrease in metabolic activity of experimental 

groups treated either with HY-PDT or specific inhibitor 17-DMAG. 

Our results confirmed inhibitory effect on CHK1 and Aurora B evoked by both therapeutic 

modalities, HY-PDT as well as 17-DMAG. Combination of both treatments may potentially 

bring enhanced effect. Therefore it will be studied in the future. 



contract No. APVV-0040-10 and the Scientific Grant Agency of the Ministry of Education 

of the Slovak Republic under the contract No. VEGA 1/0626/11. 

References 

Bagatell, R., Whitesell, L.: Altered Hsp90 function: A unique therapeutic opportunity. - In: 

Mol Cancer Ther., vol. 3, pp. 1021-1030, 2004. 

Calderwood, S.K., Khaleque, M.A., Sawyer, D.B., Ciocca, D.R.: Heat shock proteins in 


cancer: chaperones of tumorigenesis. - In: Trends in Biochemical Sciences, Vol.31, No.3: 

pp.164-172, 2006. 

Dougherty, T.J., Charles, J.G., Henderson, B.W.: Photodynamic therapy. - In Journal of the 

National Cancer Institute., Vol. 90, No. 12: pp. 889-905, 1998. 

Li, Y., Zhang, D., Xu, J., Shi, J., Jiang, L., Yao, N., Ye, W.: Discovery and development of 

natural heat shock protein 90 inhibitors in cancer treatment. – In: Acta Pharmaceutica 

Sinica B, 2(3): pp. 238–245, 2012. 

Mahalingam, D., Sword, R., Carew, J.S., Nawrocki, S.T., Bhalla, K., Giles, F.J.: Targeting 

HSP90 for cancer therapy. - In: Br J Cancer; 100 (10): pp.1523-1529, 2009. 

P4. EFFICIENCY AND TRANSPORT OF TAXANES IN HUMAN BREAST CANCER CELLS 

Marie Ehrlichova 1 , Radka Vaclavikova 1 , Katerina Kloudova 1,2 , Veronika Brynychova 1,2 , 

Vlasta Nemcova 3 , Jana Voborilova 3 , Stanislav Horsky 1 , Iwao Ojima 4 , Ivan Gut 1 and Pavel 

Soucek 1 

1 

Laboratory of Toxicogenomics, National Institute of Public Health in Prague, Czech 

Republic; ehrlichova@szu.cz 

2 

3 rd Faculty of Medicine, Charles University, Prague, Czech Republic; 

3 

Department of Cell and Molecular Biology, 3 rd Faculty of Medicine, Charles University, 

Prague, Czech Republic; 

4 

Institute of Chemical Biology & Drug Discovery, State University of New York at Stony 

Brook, Stony Brook, New York, 11794-3400, USA 

As breast cancer is the most common cancer in women worldwide, infallible methods 

for diagnosis and treatment are needed. Mitotic poisons taxanes are among the most 

successfully used drugs in chemotherapy of breast cancer. Taxanes bind to microtubules 

and cause degradation of mitotic spindle and prevent cell division. However, inherited or 

acquired multidrug drug resistance (MDR) of tumor cells may occur. MDR is connected 

with changes in expression levels of ABC and SLC transporters, biotransformation of 

xenobiotic compounds and cell cycle modifications, too. Due to MDR, the effectiveness 

of breast cancer chemotherapy is decreased or even dramatically suppressed. The aim 

of this project was to explore, if the success rate of breast cancer treatment by taxanes 

can be enhanced by the use of novel synthetic derivatives – second generation taxanes 

and fluorinated taxanes. 

Efficiency of classical, second generation and fluorinated taxanes was investigated. The 

experiment was carried out with the use of human breast cancer cell lines MDA-MB-231 

(taxane-sensitive) and MT-3 (taxane-resistant). First, cytotoxicity was measured by the 

use of MTT kit and LC50 values were determined. HPLC analysis and liquid scintillation 

were performed to show transport of paclitaxel (classical taxane) and some fluorinated 

taxanes. Next, flow cytometer BD FACSVersa was used to explore changes in cell cycle 

progression and to detect induction of cell death after incubation with taxanes. 

The effectiveness of classical and novel taxanes in sensitive breast cancer cells was similar, 

while it differed in resistant cells. Cytotoxicity test revealed that there was no observable 


difference in the effect caused either by paclitaxel or fluorinated taxanes in MDA-MB-231 

cells. On the other hand, novel taxanes were significantly more toxic for taxane-resistant 

MT-3 cells than paclitaxel. Accumulation of paclitaxel and fluorinated taxanes in taxanesensitive 

cells was also comparable, while in resistant cells, accumulation of novel 

taxanes was higher than accumulation of paclitaxel. Changes in cell cycle progression 

induced by novel and classical taxanes were determined by flow cytometry. The drugs 

caused G2/M block in both cell lines. Higher concentration of paclitaxel was needed to 

cause the same effect in resistant cells as in the sensitive ones. The effective dose of 

paclitaxel was also higher than that of fluorinated taxanes, which was more potent in 

cell cycle disruption. Paclitaxel was also shown to induce apoptosis. Late-apoptotic and 

necrotic cells occurred 24 hours after application of the drug; the effective concentration 

was 10-times higher in resistant cells than in the sensitive cells. 

In conclusion, novel taxanes act more effectively in resistant breast cancer cells than the 

classical taxane paclitaxel. The novel taxanes are therefore new potential drugs to be 

used in breast cancer therapy of patients with multidrug resistance phenotype. 


This project is supported by grants GACR 301/09/0362 and IGA NT13679-4, NT14055-3. 

P5. INHIBITION OF XIAP AND SURVIVIN SENSITIZES HUMAN COLORECTAL 

ADENOCARCINOMA CELLS TO CYTOTOXIC EFFECT OF HYPERICIN-MEDIATED 

PHOTODYNAMIC THERAPY 

Katarína Gyurászová, Jaromír Mikeš, Ján Kovaľ, Rastislav Jendželovský, Peter Fedoročko 


Košice, Slovak Republic; katarina.gyuraszova@student.upjs.sk 

Photodynamic therapy is a minimally invasive alternative form of anticancer therapy 

characterized by selective cytotoxicity toward tumour tissue. The procedure involves 

administration of a photosensitizing agent followed by irradiation at appropriate 

wavelength (Dougherty et al., 1998). Hypericin is one of the most potent naturally 

occurring photosensitizer synthesized as the secondary metabolite of St. John’s wort 

(Hypericum perforatum L.). During irradiation with light of appropriate wavelength 

excitation of hypericin to triplet state and its subsequent relaxation back to singlet 

ground state accompanied with reactive oxygen species generation occurs. A series 

of subsequent photochemical and photobiological processes lead to apoptosis and/or 

necrosis, damage of tumour vasculature and host immune response activation (Agostinis 

et al., 2002). 

Apoptosis has been accepted as a fundamental component in the pathogenesis of 

cancer. Molecules involved in regulation of apoptotic signaling pathways represent 

potential therapeutic targets (Viktorsson et al., 2005). XIAP and survivin belong to the 

family of apoptosis inhibitor proteins (IAPs). They are frequently overexpressed in cancer 

and participate in oncogenesis by assisting in resistance to apoptotic cell death. They are 

involved in several antiapoptotic and regulatory processes, thus contribute to tumour 


cell survival, resistance to anticancer therapy and disease progression (Srinivasula and 

Ashwell, 2008). Based on these findings we considered XIAP and survivin as factors with 

potentially negative impact on efficiency of hypericin-mediated photodynamic therapy 

(HY-PDT). 

Impact of specific XIAP and survivin inhibition on human colorectal adenocarcinoma HT- 

29 cells themselves and after their exposition to HY-PDT were the primary objectives 

of presented study. In our experimental system, specific inhibitors embelin (XIAP) and 

YM155 (survivin) were applied as pre-treatment as well as post-treatment to HY-PDT. 

We observed that inhibition of XIAP and survivin resulted in significant decrease in the 

metabolic activity of HT-29 cells. Further, inhibition of XIAP and survivin in combination 

with HY-PDT decreased percentage of cells with normal mitochondrial membrane 

potential, increased the number of death cells and caused accumulation of cells in G2 

phase of cell cycle. 

Considering our results, we may suggest that application of IAPs antagonists stimulates 

HY-PDT efficiency, therefore it may contribute to a more favorable outcome than the use 

of single-modality HY-PDT. 



contract No. APVV-0040-10; the Scientific Grant Agency of the Ministry of Education of 

the Slovak Republic under the contract No. VEGA 1/0626/11. 

References 

Agostinis, P., Vantieghem, A., Merlevede, W., and de Witte, P. A.: Hypericin in cancer 

treatment: more light on the way. - International Journal of Biochemistry & Cell Biology 

34: 221-241, 2002. 

Dougherty, T. J., Gomer, C. J., Henderson, B. W., Jori, G., Kessel, D., Korbelik, M., Moan, 

J., and Peng, Q.: Photodynamic therapy. - Journal of the National Cancer Institute 90: 

889-905, 1998. 

Srinivasula, S. M., and Ashwell, J. D.: IAPs: what´s in a name? - Molecular Cell 30: 123- 

135, 2008. 

Viktorsson, K., Lewensohn, R., and Zhivotovsky, B.: Apoptotic pathways and therapy 

resistance in human malignancies. - Advances in Cancer Research 94: 143-196, 2005. 

P6. L-ASPARAGINASE STRONGLY AFFECTS BIOENERGETICS IN LEUKEMIC CELLS 

Ivana Hermanova 1 , Hana Nuskova 2 , Karel Valis 3 , Karel Fiser 1 , Sonia Fernandez 4 , Josef 

Houstěk 2 , Arkaitz Carracedo 4 , Jan Trka 1 and Julia Starkova 1 

1 

Childhood Leukaemia Investigation Prague, Department of Pediatric Hematology/ 

Oncology, 2 nd Faculty of Medicine, Charles University Prague, Czech Republic, Ivana. 

hermanova@lfmotol.cuni.cz 

2 

Department of Bioenergetics, Institute of Physiology, Academy of Sciences of the Czech 

Republic, Prague, Czech Republic 

3 

Laboratory of Molecular Structure Characterization Institute of Microbiology, Academy 

of Sciences of the Czech Republic, Prague, Czech Republic 


4 

CIC bioGUNE Technology Park of Bizkaia, Derio, Bizkaia, Spain 

L-asparaginase (L-asp) is an important component of childhood acute lymphoblastic 

leukemia (ALL) therapy. Its cytotoxic effect is based on the depletion of extracellular 

asparagine and glutamine. Leukemic cells are sensitive to this depletion due to the 

lower ability to synthesize asparagine compared to healthy cells. However, the exact 

mechanism of cytotoxic effect and resistance development remains unclear. 

We established cell lines resistant to L-asp derived from the B-cell precursor ALL REH (TEL/ 

AML1[+]; very sensitive) and NALM6 (TEL/PDGFRB[+]; medium sensitive) cells by longterm 

incubation with L-asp. We used the GEP data published by Holleman (Holleman 

et al, 2004) of ALL patients´ samples sensitive/resistant to L-Asp. Pathway analysis of 

GEP data from the cell lines and patient samples revealed that protein translation and 

glucose/lipid metabolic pathways are strongly involved in the effect of L-asp. 

Next to glucose, glutamine is the other major cellular energy source, it is also important 

for the activation of PI3K/Akt/mTOR pathway - the key regulator of translation. Since 

L-asp has also glutaminase activity we focused on the effect of L-asp treatment on 

translation and bioenergetics in leukemic cells. 

The amount of p-P70S6K expression fell in REH and NALM6 after treatment with L-asp. 

Therefore we were interested whether PI3K/mTOR inhibition affects the sensitivity 

of resistant cell lines. We measured the cytostatic effect of co-treatment: L-asp with 

mTORC1 inhibitor (rapamycin); with dual inhibitor of mTORC1 and PI3K (LY294002); and 

with specific inhibitor of PI3K (PX866) on REH, res-REH, NALM6 and res-NALM6. The 

MTS assay proved the L-asp/rapamycin combination is the most effective. It strongly 

diminished viability of all cell lines. The effect was even more eminent in resistant cells. 

Next we determined the impact of L-asp on metabolism of leukemic cells. The 

protein level of c-myc, the activator of glucose and glutamine catabolism, and glucose 

transporter 1 (Glut-1) was decreased in REH and NALM6 after L-asp treatment. The 

inhibition of glycolysis was confirmed by decrease of glucose uptake and decrease of 

lactate production in cells treated with L-asp. 

Beside glutamine and glucose metabolism, fatty acids are next substrates for ATP 

production. Fatty acid oxidation (FAO) was significantly increased in both cell lines after 

L-asp. The capacity of cell respiration was also increased. 

Our results indicate that leukemic cells, which normally depend on glycolysis, are able 

to switch to fatty acid oxidation followed by activation of respiratory chain under amino 

acid deprivation stress. Respiration of healthy lymphocytes was not changed after 

incubation with L-asp. 

L-asp inhibits mTORC1, glycolysis and activates FAO and respiration. We believe that 

these changes are mainly caused by the effort of the cells to mobilize metabolism with the 

aim to supply depleted amino acids. We conclude from these data that resistance of the 

cells is caused by better biochemical adaptability to the nutrient deprived environment. 


This work was supported by IGA NT/12429-5, UNCE204012, GAUK 632513 

References 

Holleman A, Cheok MH, den Boer ML, et al.: Gene-expression patterns in drug-resistant acute 

lymphoblastic leukemia cells and response to treatment. N Engl J Med. 2004;351:533–542 


P7. MOLECULAR MECHANISMS RESPONSIBLE FOR DIFFERENT MODULATION OF 

THE CELL CYCLE PROGRESSION IN COLON CANCER CELLS TREATED WITH LA-12 AND 

OXALIPLATIN 

Iva Jelinkova 1,2 , Olga Vondalova Blanarova 1,2 , Jarmila Laukova 1,2 , Jirina Hofmanova 1,2 , 

Petr Sova 3 , Alois Kozubik 1,2 , Alena Hyrslova Vaculova 1 

1 


Republic, v.v.i., Kralovopolska 135, 612 65 Brno, Czech Republic, 

2 

Institute of Experimental Biology, Faculty of Science, Masaryk University, Terezy 

Novakove 64, 621 00 Brno, Czech Republic, 

3 

Platinum Pharmaceuticals a.s., Brno, Czech Republic 

ivina@ibp.cz, vaculova@ibp.cz 

Platinum-based antitumor agents such as oxaliplatin are used in the therapy of many 

solid cancers including colorectum, but their use is limited by serious side effects and 

intrinsic or acquired resistance. Therefore, an intensive search for novel more suitable 

candidates is still ongoing. Recently, platinum (IV) adamantylamine ligand-containing 

complex LA-12 has been introduced and shown as highly effective in many cancer cells 

including cisplatin resistant. Platinum-based drugs express their cytotoxicity by creating 

adducts on DNA that may result in initiation of DNA damage pathways. Activation of 

these pathways leads to halting the progression of the cell cycle, providing the time 

for DNA repair or induction of cell death. The key downstream target of DNA damage 

signaling pathways is p53 protein, which is essential for the cell cycle arrest, apoptosis 

and DNA repair. As a transcriptional target of p53, p21 protein is an important regulator 

of cyclins and cyclin dependent kinases. 

In our study, we compared the ability of oxaliplatin and LA-12 to modulate cell cycle and 

induce cell death in colon carcinoma cell lines HCT116 and RKO, and investigated the 

molecular mechanisms responsible for the differences in their response. We observed 

that LA-12 exerted cytotoxicity independently on p53 and p21 status, and in significantly 

lower concentration than oxaliplatin. Using p53/p21 deficient HCT116 cells, we also 

confirmed the indispensable role of these regulators in oxaliplatin-induced G2 arrest 

that was accompanied by downregulation of cyclin B1 and active Cdk1 (Flow cytometry, 

western blotting). On the other hand, LA-12-induced toxicity was not associated with 

p53- and p21-dependent G2-phase arrest and block in M phase entry observed in 

oxaliplatin-treated cells. The higher cytotoxicity together with its ability to bypass the 

cell cycle arrest important for the cellular damage repair suggest LA-12 as more effective 

candidate for elimination of colon tumors with various genetic backgrounds when 

compared to oxaliplatin. 

This work was supported by IGA Ministry of Health of the Czech Republic NT 11201- 

5, Czech Science Foundation P301/11/1730 and European Regional Development Fund 

(CZ.1.07/2.3.00/20.0180). 


P8. PROADIFEN (SKF-525A) ATTENUATES CISPLATIN RESISTANCE IN A2780CIS 

OVARIAN CARCINOMA CELLS 

Rastislav Jendželovský, Zuzana Papčová, Lucia Hiľovská, Jaromír Mikeš, Ján Kovaľ, Peter 

Fedoročko 


Košice, Slovak Republic; rastislav.jendzelovsky@upjs.sk 

Cytochrome P450 monooxygenases represent a large family of proteins involved primarily 

in phase I metabolism of xenobiotics. Some of these enzymes catalyze the conversion of 

arachidonic acid to epoxyeicosatrienoic acids (EETs), hydroxyeicosatrienoic acids (HETEs) 

and diols. Proadifen is known as a broad-spectrum inhibitor of P450 monooxygenases. 

Because of this ability proadifen should be included in the large class of non-steroidal 

anti-inflammatory drugs (NSAIDs) together with inhibitors of cyclooxygenases and 

lipoxygenases. Analogous to most of these NSAIDs proadifen possess also antiproliferative 

and pro-apoptotic activities in cancer cells originating from different tissues 

(Hoferová et al., 2004; Jendželovský et al., 2012). 

In our previous study, we have found that pre-treatment of HT-29 colon adenocarcinoma 

cells with proadifen administered prior to hypericin-mediated photodynamic therapy 

(HY-PDT) increased hypericin content and enhanced oxidative stress in the cells, which 

led to the onset of apoptosis. We also found that proadifen as P450 monooxygenase 

inhibitor affected the activity of transport proteins MRP1 and BCRP (Jendželovský et 

al., 2009). Based on these results we asked, whether proadifen is able to increase the 

toxicity of well-known chemotherapeutic agent, cisplatin, whose anticancer effect may 

be affected among other mechanisms either by enhanced efflux or increased inactivation 

(Materna et al., 2005; Wang et al., 2013). 

In this study IC20 values of proadifen in combination with IC20 values of cisplatin were 

used to treat both cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) ovarian 

carcinoma cells. Estimated IC20 values were extrapolated from an exponential (cisplatin) 

and polynomial fit (proadifen) to the metabolic activity data. Metabolic activity was 

established by MTT assay. Expression of drug efflux transporters (MRP1, MRP2, BCRP), 

CYP3A4 and proteins engaged in apoptosis and/or tumour progression was analysed by 

Western blot. Phosphatidylserine externalization and cell viability analysis, mitochondrial 

membrane depolarization, histone H2AX phosphorylation (pS139) and transport activity 

of ABC proteins were examined by flow cytometry. 

Combined proadifen and cisplatin treatment inhibited metabolic activity of A2780cis 

cells, which was accompanied by significant onset of cell death. Moreover, flow cytometry 

analyses revealed that proadifen affected mainly the function of drug efflux protein 

MRP2 but also the MRP1 in smaller extend. In agreement with these results, histone 

H2AX phosphorylation was also reversed by proadifen pre-treatment. Downregulation 

of MRP1, MRP2, CYP3A4 and anti-apoptotic proteins with most pronounced effect on 

survivin expression was also observed. 

Our findings demonstrate that downregulation of MRP2 and survivin by proadifen 

should be primarily responsible for the enhanced cytotoxic effect of cisplatin in A2780cis 


carcinoma cells. Since proadifen is a P450 monooxygenase inhibitor, the possibility that 

cisplatin effect is influenced by drug-metabolizing enzymes could not be excluded. We 

hypothesize that proadifen may be used as a strategy for the sensitization of resistant 

ovarian carcinomas to cisplatin treatment. However, future studies will be needed to 

establish the impact of proadifen on cisplatin action in vivo. 



contract Nos. APVV-0040-10 and VVCE-0001-07 and the Scientific Grant Agency of the 


References 

Hoferová, Z., Souček, K., Hofmanová, J., Hofer, M., Chramostová, K., Fedoročko, P., and 

Kozubík, A.: In vitro proliferation of fibrosarcoma cells depends on intact functions of 

lipoxygenases and cytochrome P-450-monooxygenase. - Cancer Investigation 22: 234- 

247, 2004. 

Jendželovský, R., Kovaľ, J., Mikeš, J., Papčová, Z., Plšíková, J., and Fedoročko, P.: Inhibition 

of GSK-3β reverses the pro-apoptotic effect of proadifen (SKF-525A) in HT-29 colon 

adenocarcinoma cells. - Toxicology in Vitro 26: 775-782, 2012. 

Jendželovský, R., Mikeš, J., Kovaľ, J., Souček, K., Procházková, J., Kello, M., Sačková, 

V., Hofmanová, J., Kozubík, A., and Fedoročko, P.: Drug efflux transporters, MRP1 

and BCRP, affect the outcome of hypericin-mediated photodynamic therapy in HT-29 

adenocarcinoma cells. - Photochemical & Photobiological Sciences 8: 1716-1723, 2009. 

Materna, V., Liedert, V., Thomale, J., and Lage, H.: Protection of platinum-DNA adduct 

formation and reversal of cisplatin resistance by anti-MRP2 hammerhead ribozymes in 

human cancer cells. - International Journal of Cancer 115: 393-402, 2005. 

Wang, H., Li, X., Chen, T., Wang, W., Liu, Q., Li, H., Yi, J., and Wang, J.: Mechanisms of 

verapamil-enhanced chemosensitivity of gallbladder cancer cells to platinum drugs: 

glutathione reduction and MRP1 downregulation. - Oncology Reports 29: 676-684, 2013. 

P9. MITOCHONDRIAL STATUS OF MHTT MINIATURE PIG SPERMATOZOA DETERMINED 

BY FLOWCYTOMETRY 

Jiri Klima, Ivona Valekova, Monika Macakova, Antonin Pavlok, Jan Motlik 

Institute of Animal Physiology and Genetics, Academy of Sciences of Czech Republic, 

Libechov, Czech Republic; klima@iapg.cas.cz 

Huntington´s disease (HD) is caused by CAG repeat expansion in huntingtin gene. Yet 

uncharacterized „gain of function“ of mutant huntingtin (mHtt) is responsible for this 

devastating disorder affecting not only particular neural cell populations of central neural 

system but also other cells, tissues and organs. MHtt affects various cellular processes 

among them are mitochondrial biogenesis, mitophagy and metabolism. Moreover, 

mitochondria are involved in mHtt driven adverse effects like excitotoxicity or elevated 

formation of reactive oxygen species (ROS). 

Reproductive defects have also been documented both in HD patients and small animal 


models. As manifested by compromised fertilization parameters, some transgenic boars 

of miniature pig line bearing mutated N-terminal part of human huntingtin produce 

low quality semen too. Functional spermatozoa mitochondrion plays a substantial role 

in oocyte fertilization, therefore the impact of mHtt expression on semen quality and 

spermatozoa parameters had been determined using flowcytometry approach. 

The mitochondrial mass was estimated by nonyl Acridine Orange (NAO) staining of 

cardiolipin. NAO signals do not differ between transgenic (Tg) and wild type (Wt) 

spermatozoa. Although defective in motility Tg spermatozoa displayed mitochondrial 

membrane potential similar to Wt as assessed by JC-1 staining. 

Both Tg and Wt spermatozoa produce ROS spontaneously as determined by MitoSOX TM 

Red (MSR) reagent. While there is no difference in maximal levels of MSR signal measured 

between Wt and Tg animals, higher ROS production is indicated by sooner development 

of detectable MSR signal during Tg spermatozoa staining procedure compared to Wt 

spermatozoa. 

Acknowledgement 

This work was supported by the CHDI Foundation (ID 1035, A5378), The Technical 

Agency of the Czech Republic (TA01011466), RVO 67985904, and European Regional 

Development Fund CZ.1.05/2.1.00/03.0124. 

P10. THE CELL DEATH INDUCED BY TAXANES IN PANCREATIC CANCER CELL LINES 

Katerina Kloudova 1,2 , Marie Ehrlichova 1 , Radka Vaclavikova 1 , Veronika Brynychova 1,2 , 

Martin Oliverius 3 , Eva Honsova 3 , Matej Kocik 3 , Iwao Ojima 4 and Pavel Soucek 1 

1 

Laboratories of Toxicogenomics, National Institute of Public Health in Prague, Czech 

Republic; katerina.kloudova@szu.cz 

2 

3 rd Faculty of Medicine, Charles University in Prague, Czech Republic; 

3 

Institute for Clinical and Experimental Medicine, Prague, Czech Republic; 

4 

Institute of Chemical Biology & Drug Discovery and Department of Chemistry, State 

University of New York at Stony Brook, New York, USA. 

Pancreatic carcinoma is one of the most aggressive tumours with high mortality 

worldwide (Jemal et al., 2007). Gemcitabine is primary cytotoxic agent used in the 

therapy of pancreatic carcinoma. Unfortunately, treatment with gemcitabine has so far 

proved ineffective (Hildago, 2010). Combined therapy together with taxane presents 

effort to increase of antitumor effect of gemcitabine (Frese et al., 2012). Taxanes are 

successfully used in therapy of various human cancers, mainly of breast and ovarian 

carcinomas. However, inherited or acquired resistance of tumour cells to taxanes 

(paclitaxel, docetaxel) presents one of the major obstacles in successful therapy. Drug 

resistance is a multifactorial process that may be related to drug transport, metabolism 

or alterations in apoptosis induction by e.g. taxanes. Certain novel taxanes (i.e. taxanes 

of second generation or fluorinated taxanes) possess extremely high potency against 

drug-resistant cancer cells expressing the multidrug resistance (MDR) phenotype (Ojima 

et al., 1996; Ehrlichova et al., 2012) The aim of our study was to investigate the effect 


of classical and novel taxanes on cell death and cell cycle in pancreatic cancer cell lines. 

Our experimental model is composed of three tumour pancreatic cell lines; gemcitabine 

sensitive BxPC-3, resistant MIAPaCa-2 and cell line Paca-44. These cell lines also differ in 

mutational spectra of KRAS gene (Paca-44 and MIAPaCa-2 mutated vs. BXPc-3 wild type). 

Cytotoxicity of cell lines to taxanes (paclitaxel, SB-T-1214 and SB-T-1216) was measured 

by MTT. Modulation of cell cycle and induction of cell death by taxanes were followed 

by flow cytometry. In addition, gene expression of selected ABC transporters known to 

transport taxanes was quantified using real-time PCR with relative quantification. 

Pancreatic carcinoma cell lines Paca-44 and BxPC-3 were more sensitive than MIAPaCa-2 

to all examined taxanes (2-3-times). In general, classical taxane paclitaxel was more toxic 

than SB-T taxanes in all three cell lines. The effective concentrations for induction of G2/M 

block in pancreatic cells by taxanes were 100nM for paclitaxel and SB-T-1214 in Paca- 

44 and MIAPaCa-2 cells. Novel taxane SB-T-1216 was efficient in 100nM concentration 

in Paca-44 cells and 300nM in MIAPaCa-2. Furthermore, very low concentrations of 

paclitaxel (10nM), SB-T-1214, SB-T-1216 (30nM) induced subpeak G0/G1 in Paca-44 

cell. The induction of apoptosis by taxanes was measured by Annexin V assay kit in all 

three cell lines. In addition, gene expression profile of ABC transporters (ABCA1, ABCA3, 

ABCA7, ABCB1, ABCB2, ABCB3, ABCB4, ABCB11, ABCC1, ABCC2, ABCC3, ABCC5, ABCC5, 

ABCC6, ABCC7, ABCC8, ABCC10, ABCG1, ABCG2) was estimated. ABCC genes exerted 

higher expression levels than members of ABCB family in all cell lines used. Cells differed 

in gene expression of ABCA3, ABCB11, ABCC2, ABCC6, ABCG1 and ABCG2. All three cell 

lines did not expressed ABCB1 gene at all. 

In conclusion, Paca-44, MIAPaCa-2, BxPC-3 pancreatic cell lines seem to be very good 

experimental models for study of classical and novel taxane efficiency. These different 

types of pancreatic cell lines have variable sensitivity to classical and promising novel 

taxane analogs suggesting potential use in resistant types of solid tumours. 


This work was supported by grants GACR P301/12/1734 and IGA NT/14055-3. 

References 

Ehrlichova, M., Ojima, I., Chen, J., Vaclavikova, R., Nemcova-Furstova, V., Voborilova, J., 

Simek, P., Horsky, S., Soucek, P., Kovar, J., Brabec, M., and Gut, I.: Transport, metabolism, 

cytotoxicity and effects of novel taxanes on the cell cycle in MDA-MB-435 and NCI/ADR- 

RES cells. - Naunyn Schmiedebergs Arch Pharmacol. 385: 1035-1048, 2012. 

Frese, K.K., Neesse, A., Cook, N., Bapiro, T.E., Lolkema, M.P., Jodrell, D.I., and Tuveson, 

D.A.: nab-Paclitaxel potentiates gemcitabine activity by reducing cytidine deaminase 

levels in a mouse model of pancreatic cancer. - Cancer Discovery 2: 260-269, 2012. 

Hidalgo M.: Pancreatic cancer. - N Engl J Med 362: 1605–1617, 2010. 

Jemal, A., Siegel, R., Ward, E., Murray, T., Xu, J., and Thun, M.J.: Cancer statistics, 2007. - 

CA Cancer J Clin 57: 43-66, 2007. 

Ojima, I., Slater, J.C., Michaud, E., Kuduk, S.D., Bounaud, P.Y., Vrignaud, P., Bissery, M.C., 

Veith, J.M., Pera, P., and Bernacki, R.J.: Syntheses and structure-activity relationships 

of the second-generation antitumor taxoids: exceptional activity against drug-resistant 

cancer cells. - J Med Chem. 39: 3889-3896, 1996. 


P11. NEW BIOACTIVE AND FLUORESCENT MACROLIDE COMPOUND ISOLATED FROM 

STREPTOMYCES DURMITORENSIS 

Koukalova Alena 1,2 , Cerny Jan 1 , Humpolickova Jana 2 

1 

Faculty of Science, Charles University in Prague, Prague, Czech Republic, alena. 

koukalova@natur.cuni.cz 

2 

J. Heyrovsky Institute of Physical-Chemistry, Academy of Sciences of the Czech Republic, 

Prague, Czech Republic 

One possibility how to discover new bioactive compounds is to focus on screening 

various bacterial secondary metabolites, especially isolated from newly described 

species. Actinomycetes and in particular the genus Streptomyces represents very 

important source of such naturally preselected biologically active compounds. 

Some of the polyene macrolides found in bacteria are very useful antibiotics 

and antifungal agents or experimental tools (Berdy, 2005). Association between 

macrolides and particular membrane components is a controversial issue, and there 

is only rare biophysical evidence for direct interactions. The mode of interaction with 

the membrane could be a key for understanding the molecular mechanism 

of bioactivity. 

We are thoroughly characterizing a new polyene macrolide 

32,33-didehydroroflamycoin (DDHR) produced by the bacteria Streptomyces 

durmitorensis found in soil samples in Montenegro (Savic et al., 2007) related to the 

macrolide filipin and closely related to the antibiotic roflamycoin (Stodulkova et al., 

2011). Previous data suggested that DDHR has an ability to rearrange actin and tubulin 

cytoskeleton in treated cells and induce apoptosis (Stodulkova et al. 2011). 

On the contrary to published results we have found out that DDHR causes necrosis rather 

than apoptosis. Mode of toxicity was quantified by using flow cytometry (Annexin-V/ 

DAPI) and confirmed by DNA electrophoresis. 

DDHR has a bioactivity and toxicity to various cell types. Prokaryotic cells represented 

by Escherichia coli are not sensitive. On contrary, yeast Saccharomyces cerevisiae and 

various types of human cells respond to micromolar concentrations. Biological activity 

assay performed on HeLa cells, T-lymphocyte cell line and primary skin fibroblasts has 

shown dissimilar toxic effect of DDHR to the different types of mammalian cells (IC 50 

= 25 

µM for Hela cells, 15 µM for primary skin fibroblasts and T-lymphocyte cell line). 

DDHR is not only a bioactive molecule, but also demonstrates interesting fluorescence 

properties, which allows visualization of particular internal membrane structures even 

at very low (

The pore-forming activity of DDHR was investigated on individual giant unilamellar 

phospholipid vesicles (GUVs) made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) 

and DOPC with 30% content of cholesterol. Using multiphoton fluorescence microscopy 

we observed formations of necklace-shaped structures or internal small particles inside 

GUVs made of DOPC. This behaviour is consistent with the theory of pore formation 

as mode of action explaining the bioactivity of some particular secondary metabolites 

(Zemel at al., 2005). Content of cholesterol in GUVs inhibits formation of these structures. 

Permeability of membranes after treatment with DDHR was proved in the standard dye 

leakage assay performed on the same vesicle types. 

We have also proved the direct interaction between cholesterol and DDHR using 

FTIR spectroscopy. This interaction could play a crucial role in its bioactivity and 

the ability to stain only certain membrane components in cells. 


This work was supported by Long-Term Research Plans of the Ministry of Education, 

Youth and Sports of the Czech Republic No. MSM0021620858. Author is thankful to 

the laboratory of RNDr. Miroslav Flieger, CSc., Institute of Microbiology, Academy 

of Sciences of the Czech Republic. 

References 

Berdy, J. (2005). Bioactive Microbial Metabolites. The Journal of Antibiotics, 58(1): 

1-26. 

Savic, M., Bratic, I., & Vasiljevic, B. (2007). Streptomyces durmitorensis sp. nov., a 

producer of an FK506-like immunosuppressant. International journal of systematic and 

evolutionary microbiology, 57(Pt 9): 2119-24. 

Stodulková, E., Kuzma, M., Hench, I. B., Černý, J., Králová, J., Novák, P., Chudíčková, 

M., et al. (2011). New polyene macrolide family produced by submerged culture of 

Streptomyces durmitorensis. The Journal of antibiotics, 64(11): 717-22. 

Zemel, A., Ben-Shaul, A., May, S. (2005). Perturbation of a lipid membrane by amphipathic 

peptides and its role in pore formation. European biophysics journal, 34(3): 230-42. 

P12. MODULATION OF HYPERICIN-MEDIATED PHOTODYNAMIC THERAPY USING 

HISTONE DEACETYLASE INHIBITORS 

Ján Kovaľ, Andrea Halaburková, Rastislav Jendželovský, Jaromír Mikeš, Peter Fedoročko 


Košice, Slovak Republic; jan.koval@upjs.sk 

Photodynamic therapy (PDT) is a minimally invasive cancer treatment method which 

utilizes different tumor-localizing agents that are activated by irradiation with light 

of a specific wavelength. This triggers production of reactive oxygen species, which 

subsequently activate processes implicated in irreversible changes to various cellular 

targets leading to cell death (Dougherty et al., 1998). Hypericin (HY), a naturally occurring 

aromatic polycyclic diantraquinone from St. John’s wort (Hypericum perforatum L.), has 

unique photocytotoxic properties suitable for PDT. 


Acetylation of histones, along with other epigenetic modifications, has been described 

as the main epigenetic regulator controlling cell fate and therefore histone deacetylase 

inhibitors (HDIs) are being investigated as a new promising group of anticancer drugs 

(De Ruijter et al., 2003). HDIs induce hyperacetylation of proteins/histones, thus 

decondensing chromatine structure, which subsequently increases the accessibility of 

DNA to transcription factors and activates specific genes, tumor suppressor, oncogenes 

and non-histones proteins implicated in differentiation, proliferation, cell cycle and 

apoptosis. Moreover, HDIs may enhance, through processes described above, cytotoxicity 

of drugs targeting DNA. HDIs are of both natural and synthetic origin and they have 

demonstrated potent anticancer activity in many pre-clinical and clinical trials, either 

as monotherapies or in combination with conventional chemotherapy (Glaser, 2007). 

These include derivates of hydroxamic acid: suberoylanilid hydroxamic acid (Vorinostat, 

SAHA - treatment of cutaneous T cell lymphoma) and trichostatin A (TSA - antifungal 

antibiotic); and a group of short-chain fatty acids HDIs: valproic acid (VPA - epilepsy drug) 

and sodium phenylbutyrate (NaPB – disorders of the urea cycle). 

Remodeling of chromatin structure may contribute to enhanced sensitivity to 

photochemical and photobiological processes caused by PDT, increasing the ability of 

oxygen radicals to attack DNA thus influence cell differentiation and cell death. The aim 

of this study was therefore to evaluate the impact of HDIs alone and in combination with 

hypericin-mediated photodynamic therapy (HY-PDT) on the response of a human HT-29 

colon adenocarcinoma cell line. 

Based on extensive screening using the MTT assay, one hypericin and two concentrations 

for each HDI were chosen for further experiments. Analysis of total cell number 

demonstrated that pre-treatment with all HDIs alone and HY-PDT reduced the total cell 

number significantly. A more pronounced drop in this parameter was observed when 

combined treatment of HDIs with HY-PDT was applied. The impact of HDIs and of HY-PDT 

was further characterized using flow cytometry. Lower concentrations of TSA significantly 

decreased mitochondrial membrane potential (MMP) in comparison to other HDIs. Pretreatment 

with higher concentrations of SAHA and TSA made cells more vulnerable to 

damage resulting from HY-PDT and this led to a considerable dissipation of the MMP. A 

similar pattern of the effect on MMP between the two groups of HDIs was also observed. 

Subsequently, we analysed the effect of HDIs, HY-PDT and their combination on the 

viability/metabolic activity of cancer cells. Similarly to MMP, we observed a higher 

potential of the hydroxamic acid derivates (SAHA, TSA) to sensitize cancer cells to the 

effect of HY-PDT. We detected a significantly higher number of dead cells in combined 

treatment than when drugs were applied alone at both time intervals. The analysis of 

cell cycle distribution revealed an accumulation of cells in the S phase after treatment 

with higher concentrations of SAHA, TSA and VPA in combination with HY-PDT. 

In this study we observed that HDIs are able to modulate and enhance the cytotoxic effects 

of HY-PDT. However, the details of molecular mechanisms mediating photodynamicsensitization 

by HDIs are at present, not elucidated. 



contract no. APVV-0040-10 and VVCE-0001-07 and the Scientific Grant Agency of the 



References 

De Ruijter, A.J.M., van Gennip, A.H., Caron, H.N., Kemp, S., and van Kuilenburg, 

A.B.: Histone deacetylases (HDACs): characterization of the classical HDAC family. – 

Biochemical Journal 370(Pt 3): 737-749, 2003. 

Dougherty, T.J., Gomer, C.J., Henderson, B.W., Jori, G., Kessel, D., Korbelik, M., Moan, J., 

and Peng, Q.: Photodynamic therapy – Journal of the National Cancer Institute 90(12): 

889-905, 1998. 

Glaser, K.B.: HDAC inhibitors: clinical update and mechanism-based potential. – 

Biochemical Pharmacology 74(5): 659-671, 2007. 

P13. MYELOPEROXIDASE DEFICIENCY ATTENUATES THE CELL DEATH OF NEUTROPHILS 

INDUCED BY OXIDATIVE BURST 

Silvie Kremserová 1,2 , Karel Souček 1,3 , Anna Klinke 4 , Hana Kolářová 1,3 , Stephan Baldus 4 , 

Jason P. Eiserich 5 , Lukáš Kubala 1,3 

1 

Institute of Biophysics, Academy of Sciences of the Czech Republic; 

kremserova.s@gmail.com 

2 

Department of Animal Physiology, Faculty of Science, Masaryk University, Czech 

Republic; 

3 

International Clinical Research Center - Center of Biomolecular and Cellular 

Engineering, St. Anne‘s University Hospital Brno, Brno, Czech Republic; 

4 

Department of Internal Medicine, School of Medicine, University of California, Davis, CA; 

5 

Heart Center, University of Cologne, Cologne, Germany 

Neutrophils play a critical role in host defense. On the other hand, neutrophils 

accumulated at the site of inflammation contribute to tissue injury associated with acute 

and chronic inflammatory disease. Thus, a neutrophil clearance by apoptosis is a key 

mechanism for the inflammation resolution. (Klebanoff, 2005, Borregaard and Cowland, 

1997). 

Myeloperoxidase (MPO) is the most abundant enzyme in granules of neutrophils that 

is release upon their activation during degranulation process. Interestingly, in mouse 

models of acute and chronic inflammation, MPO deficiency is connected with a more 

severe course of the inflammatory process (Tsurubuchi et al., 2001). This can be due to 

reduced clearance of MPO-deficient neutrophils from the site of inflammation. Thus, the 

involvement of MPO in neutrophil cell death was studied. 

Neutrophils isolated from MPO-deficient mice exhibited a significantly lower rate of cell 

death compared to neutrophils isolated from wild type mice in response to the stimulation 

of oxidative burst by the activators phorbol-12-myristate-13-acetate and ionomycin. 

Interestingly, the spontaneous cell death rate was similar for neutrophils isolated from 

MPO-deficient and wild type mice. The cell death induced by the employed activators 

was connected with a massive increase in the surface expression of phosphatidylserine 

detected by Annexin V, which was significantly reduced in neutrophils isolated from 

MPO-deficient mice. Interestingly, the activation of caspase 3 was not detected in these 


stimulated cells. The physiological importance of this phenomenon is supported by the 

observation of a significant accumulation of neutrophils in the lungs of MPO-deficient 

mice compared to wild type mice in a model of acute pulmonary inflammation induced 

by the intranasal application of lipopolysaccharide. 

Collectively, these results suggest that neutrophil-derived MPO can control the course 

of the inflammation process through the ability of MPO to modulate the life span of 

neutrophils. 


This work was supported by grants from the Czech Science Foundation No. P305/12/ 

J038 and from DFG BA1870/9-1; KL 2516/1-1 and the European Social Fund - Project 

OrganoNET (CZ.1.07/2.4.00/31.0245) and HistoPARK (CZ.1.07/2.3.00/20.0185). LK and 

KS were supported by the European Regional Development Fund - Project FNUSA-ICRC 

(No. CZ.1.05/1.1.00/02.0123). 

References 

Borregaard, N., and Cowland, J.B.: Granules of the human neutrophilic 

polymorphonuclear leukocyte. - Blood 89, 3503-3521, 1997. 

Klebanoff, S.J.: Myeloperoxidase: friend and foe. - Journal of Leukocyte Biology, 

77(5):598-625, 2005. 

Tsurubuchi, T., Aratani, Y., Maeda, N., Koyama, H.: Retardation of early-onset PMAinduced 

apoptosis in mouse neutrophils deficient in myeloperoxidase. - Journal of 

Leukocyte Biology 70(1): 52-58, 2001. 

P14. THE ROLE OF B CELLS IN THE EARLY STAGES OF IMMUNE RESPONSE AGAINST 

INTRACELLULAR BACTERIAL PATHOGEN FRANCISELLA TULARENSIS 

Zuzana Krocova, Klara Kubelkova, Lenka Plzakova, Lenka Zarybnicka, Zuzana Sinkorova, 

Ales Macela 

University of Defence, Faculty of Military Health Sciences, Hradec Kralove, Czech 

Republic 

E-mail: krocova@pmfhk.cz 

Keywords: B cells, Francisella tularensis, Innate immunity 

Francisella tularensis, as an intracellular bacterial pathogen, adhere, interact, and enter 

the range of phagocytic and non-phagocytic cells. Recently, we have demonstrated, 

using in vitro model, that both, vaccine strain F. tularensis LVS and virulent F. tularensis 

strain FSC200 are able to adhere and even to enter the human (Ramos) and mouse 

(A20) B cells where survive in non-replicative state. The entrance of F. tularensis into B 

cells required active participation of bacterium and engagement of B cell receptor. We 

provided the in vivo analysis of the cellular response during early stages of F. tularensis 

FSC200 i.p. infection on murine model. The phenotyping of leukocyte populations, B 

cell populations (B-1a, B-1b and B-2), phenotype of infected cells, the expression of 

activating markers (MHC II, CD25, CD54, CD69 CD71, CD80 and CD86) on B-1a, B-1b and 

B-2 cells and production of cytokines by B cells sorted from peritoneal cavity and spleen 


was analyzed. We found out that, in the site of infection, B-1a cells are activated very 

early after infection (within 12 hours) and in this time B cells relatively broad spectrum of 

cytokines. Thus, considering the protective efficacy of circulating antibodies, production 

of cytokines, and potent antigen-presenting function of B cells, B cell-mediated, as well 

as T cell-mediated immunity plays an equivalent role in control of F. tularensis infection 

in mice. 


The authors are supported by the grant from Grant Agency of Czech Republic No. 

P302/11/1631 

P15. SENSITIZATION OF CELLS TOWARDS ANTICANCER TREATMENT BY INHIBITORS OF 

SUCCINATE DEHYDROGENASE 

Björn Kruspig 1 , Belma Skender 2,3 , Boris Zhivotovsky 1 , and Vladimir Gogvadze 1 

1 

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 

210, Stockholm, SE-171 77 Sweden 

2 


Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic 

3 

Department of Animal Physiology and Immunology, Institute of Experimental Biology, 

Faculty of Science, Masaryk University, Terezy Novákové 64, 621 00 Brno, Czech 

Republic 

Cancer and apoptosis are regarded as antagonistic processes. Apoptosis may be 

involved in spontaneous regression of tumors, whereas defects in apoptotic pathways 

may contribute to tumor progression and resistance to treatment. Therefore, the 

stimulation of cell death is the most widely used tools in cancer therapy. Considering 

mitochondria as key participants in various forms of cell death steps directed to 

mitochondrial destabilization, permeabilization of the outer mitochondrial membrane 

(OMM) and release of pro-apoptotic proteins represent a promising strategy in fighting 

cancer. Mitochondria are multifunctional organelles, playing different vital roles in 

cell metabolism, survival and death. One of their essential physiological functions is 

the generation of ATP for metabolic demands through the oxidative phosphorylation 

(OXPHOS) pathway. They are also known as a major site of reactive oxygen species (ROS) 

production, which participates in different signaling pathways, but might cause cell 

damage if produced excessively. In addition, mitochondria are involved in intracellular 

Ca 2+ homeostasis regulation. All these functions of mitochondria are essential for cell 

survival and also play an important role in cell death stimulation, thus making these 

organelles an attractive target for cancer therapy. 

In the present work we analyzed possible consequences of combined treatment of 

tumor cells with inhibitors of succinate dehydrogenase (complex II of the mitochondrial 

respiratory chain) – thenoyltrifluoroacetone (TTFA) and methyl-malonate (MM) – in 

combination with the conventionally used anticancer drug cisplatin. 

Incubation of neuroblastoma Tet21N and SK-N-BE(2) cells with 5 µg/ml cisplatin for 24 


h induced very minor manifestations of apoptosis, including morphological changes, 

release of cytochrome c from mitochondria, stimulation of caspase 3-like activity, and 

cleavage of poly (ADP-ribose) polymerase (PARP). However, in a combined treatment with 

cisplatin and 100 µM TTFA a strong increase of the drug-induced toxicity was observed, 

whereas TTFA by itself did not cause any toxicity. Measuring mitochondrially produced 

ROS with a superoxide-sensitive dye that is specifically targeted to the mitochondria, 

revealed a strong induction of ROS by TTFA in combination with cisplatin. Antioxidant 

N-acetyl-cysteine (NAC) partially reversed the stimulatory effect of TTFA on cisplatininduced 

apoptosis. Both results clearly demonstrate the importance of ROS produced by 

complex II for cellular response to anticancer treatment. 

Another inhibitor of complex II, methyl-malonate, failed to stimulate cisplatin-induced 

apoptosis. Interestingly, both drugs convey their effect by inhibiting different subunits 

of the succinate dehydrogenase complex, indicating an importance of the activity of 

individual subunits for the sensitivity of cells to drug induced cell death. 

P16. INTERACTION OF MYELOPEROXIDASE WITH ENDOTHELIAL CELLS 

Hana Kolářová 1,2 , Jan Víteček 1,2 , Silvie Kremserová 1,3 , Anna Klinke 4 , Stephan Baldus 4 , 

Lukáš Kubala 1,2 

1 

Institute of Biophysics, Academy of Sciences of the Czech Republic; 

2 


Engineering, St. Anne‘s University Hospital Brno, Brno, Czech Republic; 

3 

Department of Animal Physiology, Faculty of Science, Masaryk University, Czech 

Republic; 

4 

Heart Center, University of Cologne, Cologne, Germany 

Activation of polymorphonuclear neutrophils (PMN) and release of myeloperoxidase 

(MPO), expressed in large amounts in PMN, are suggested to be important in the vascular 

inflammatory processes. It was shown that MPO can bind to vascular endothelium and 

undergo transcytosis. However, the importance of the interaction of highly cationic MPO 

with glycocalyx on endothelial surface layer, which is composed of glycosaminoglycans 

(GAGs) and proteoglycans, for the glycocalyx integrity and function has not been 

described. Herein we focused on characterization of MPO interaction with selected GAGs 

and proteins that can be found in extracellular matrix produced by vascular endothelium. 

We show that MPO binds to the ECM proteins collagen IV and fibronectin, and 

this association is enhanced by the pre-incubation of these proteins with GAGs. 

Correspondingly, an excess of GAGs in solution during incubation inhibits the binding of 

MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived 

ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to 

collagen IV and fibronectin; even the potentiation of MPO activity in the presence of 

collagen IV and fibronectin was observed. 

Further, we aimed to characterize to which extent MPO modulates the charge and the 

three dimensional structure of the glycocalyx. The hypothesis of the project assumes 


that the interaction of MPO with glycocalyx GAGs can lead to collapse of the glycocalyx 

structure. In order to get an insight into the mechanism how MPO may affect this, MPO 

mediated modulation of viscosity of GAGs solution as a degree of modulation of GAG 

three dimensional structure was determined. Data show a remarkable MPO mediated 

increase of the viscosity of two model GAGs hyaluronate and chondroitin sulphate. 

Further, MPO mediated decrease of GAG intrinsic negative charge at physiological 

conditions was evaluated. Different experimental settings were tested to stabilize GAGs 

in three dimensional structures which allowed the determination of the charge change 

employing dyes alcian blue X8 or direct red. Results suggest that staining with both dyes 

corresponds in a linear manner to the amount of embedded GAGs and that cationic 

proteins decrease the overall charge of embedded GAGs. Interestingly, similar effect was 

observed in vivo in mouse after application of alcian blue into the carotid artery and 

consequent loss of staining after application of MPO. 

These findings extend the knowledge of MPO inflammatory properties in vascular 

inflammation. 


This work was supported by grants from the Czech Science Foundation No. P305/12/ 

J038 and from DFG BA1870/9-1; KL 2516/1-1 and the European Social Fund - Project 

OganoNET (CZ.1.07/2.4.00/31.0245) and HistoPARK (CZ.1.07/2.3.00/20.0185). LK was 

supported by the European Regional Development Fund - Project FNUSA-ICRC (No. 

CZ.1.05/1.1.00/02.0123). 

P17. ROSIGLITAZONE ACTS AS AN EFFICIENT MODULATOR OF LA-12-INDUCED 

CYTOTOXIC AND CYTOSTATIC RESPONSE OF COLON CANCER CELLS 

Jarmila Lauková 1,2,3 , Iva Jelínková 1,2 , Jiřina Hofmanová 1,2 , Nicol Straková 1,6 , Petr Sova 4 , Jiří 

Ehrmann 5 , Zdeněk Kolář 5 , Alois Kozubík 1,2 and Alena Hyršlová Vaculová 1,3 

1 


Republic, v.v..i., Brno, Czech Republic; laukova@ibp.cz, vaculova@ibp.cz 

2 

Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech 

Republic 

3 


St. Anne´s University Hospital Brno, Czech Republic 

4 

Platinum Pharmaceuticals, a.s., Brno, Czech Republic 

5 

Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, 

Palacky University Olomouc, Czech Republic 

Rosiglitazone, a synthetic ligand of peroxisome proliferator-activated receptor gamma 

(PPARgamma) can suppress proliferation and stimulate death of colon cancer cells. 

In addition, it may also act as an agent sensitizing the cancer cells to the action of 

selected chemotherapeutic drugs, which suggests its potential application in antitumor 

therapy. Our results showed that pretreatment of HCT116 human colon cancer cells 

with rosiglitazone resulted in enhancement of apoptosis induced by platinum (IV) 


chemotherapeutic drug LA-12. These effects were caspase-dependent, involved 

mitochondrial apoptotic pathway, and occurred independently on p53. 

Here we investigated the relationship between the apoptosis induction and the cell cycle 

modulation following the combined treatment of colon cancer cells with rosiglitazone 

and LA-12. The particular attention was paid to the S- and G2/M-related changes, 

and associated regulatory proteins (e.g. cyclins and cyclin-dependent kinases). The 

functional role of the molecules involved in the cell cycle and DNA damage response 

regulation in the combined cytotoxic/cytostatic action of rosiglitazone and LA-12 was 

examined using the specific colon cancer cell lines with their deficiency/silencing, and 

flow cytometry-based analyses. In addition, the involvement of selected kinases (MAPK, 

Akt) in modulation of apoptotosis induced by the drug combination was investigated, 

and these new findings will be presented in our contribution. 

Our results suggest that rosiglitazone can positively modulate LA-12-induced cytotoxic 

and cytostatic response of colon cancer cells, which highlights its possible therapeutic 

application in this tumor type. 


This work was supported by the IGA of the Ministry of Health of the Czech Republic (NT 

11201-5), Czech Science Foundation P301/11/1730 and FNUSA-ICRC European Regional 

Development Fund No. CZ.1.05/1.1.00/02.0123, HistoPARK - CZ.1.07/2.3.00/20.0185 

P18. THE SIGNIFICANCE OF HYPOXIA AND HIF-1ΑLFA FOR THE DEVELOPMENT OF 

CHEMORESISTANCE IN NEUROBLASTOMA CELL LINES 

Marikova H. 1 , Hrabeta J. 1 , Groh T. 1,2 , Cipro Š. 1,3 , Rahman M. A. 1 , Eckschlager T. 1 

1 

Department of Paediatric Hematology and Oncology, 2 nd Faculty of Medicine, Charles 

University in Prague and University Hospital Motol, Czech Republic; h.marikova@gmail. 

com 

2 

Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech 

Republic; 

3 

Department of Pathology and Molecular Medicine, 2 nd Faculty of Medicine, Charles 

University in Prague and University Hospital Motol, Czech Republic 

Hypoxia is the hallmark of the solid tumour microenvironment. Adaptation of tumour cells 

to hypoxic conditions has important biological effects and contributes to the aggressive 

attitude of tumours and their dedifferentiation. Hypoxia is one of the reasons for reduced 

apoptosis in cytostatic-treated cancer cells and increases their genetic instability. The 

main factor necessary for adaptation to hypoxic environment is hypoxia-inducible 

transcription factor (HIF). Based not only on our previous work, the inhibition of HIF- 

1α can suppress the chemoresistance of hypoxic tumour cells to various antineoplastic 

agents, but only partially. Therefore, it is still discussed whether the chemoresistance 

of tumor cells is induced by hypoxia itself or whether it is due to the expression of 

HIF-1α or also another factor. Beside HIF-1α, it seems that other factors also cause 

chemoresistance. 


In our work, we focus on exploring the other factors involved in hypoxia-induced 

chemoresistance, both dependent and independent on HIF-1α. We have supressed the 

expression of HIF-1α in UKF-NB-4 cell line by shRNA gene silencing. In this cell line, we 

observed lower hypoxia induced chemoresistance to cisplatin, but not its absolutely 

withdrawal, which is consistent with our previous results, when we used small-molecule 

HIF-1α inhibotors and siRNA gene silencing. We started to work with hypoxia-conditioned 

media and observed that the chemoresistance is still present also in cells incubated with 

media coming from the HIF-1α knockdown cell line. We also focused on a research on 

V-ATPase and its impact on chemoresistance. 

The research results will contribute to the understanding of the biological properties of 

neuroectodermal tumors, especially the hypoxia-induced resistance to cytostatics. 


This work is supported by GA UK 620612. 

References 

Nishisho, T., Hata, K., Nakanishi, M., et al.: The a3 Isoform Vacuolar Type H+-ATPase 

Promotes Distant Metastasis in the Mouse B16 Melanoma Cells. - Mol Cancer Res. 

2011;9(7):845-55. 

Schnitzer, S. E., Schmid, T., Zhou, J., et al.: Hypoxia and HIF-1alpha protect A549 cells 

from drug-induced apoptosis. - Cell Death Differ. 2006;13(9):1611-3. 

Semenza, G. L.: HIF-1: upstream and downstream of cancer metabolism. Curr Opin 

Genet Dev. 2010 Feb; 20(1):51-6. Epub 2009 Nov 26. 

Yee Koh, M., et al.: HIF-1 regulation: not so easy come, easy go. - Trends Biochem Sci. 

2008 Nov; 33(11):526-34. Epub 2008 Sep 21. 

P19. EVALUATION OF CHEMOPREVENTIVE ACTIVITY OF PHYTOCHEMICALS USING 

NORMAL AND TUMORIGENIC RAT LIVER EPITHELIAL CELLS 

Premysl Mikula 1 , Zuzana Lencesova 1,2 , James Trosko 3 , Brad Upham 3 , Pavel Babica 1,2 

1 

Institute of Botany of the ASCR, Department of Experimental Phycology and 

Ecotoxicology, Lidicka 25/27, 602 00 Brno, Czech Republic; premysl.mikula@centrum.cz 

2 

Masaryk University, RECETOX – Research Centre for Toxic Compounds in the Environment, 

Kamenice 753/5, 625 00 Brno, Czech Republic 

3 

Michigan State University, Department of Pediatrics and Human Development, East 

Lansing, MI 48824, USA 

Cancer chemopreventive and anticancer potential of selected phytochemicals (e.g. 

metformin, silibinin and quercetin) was evaluated in WB-F344 rat liver epithelial cells as 

well as in WB-ras cell line derived from WB-F344 cells by stable transduction with H-ras 

oncogene. 

While WB-F344 cell line represents normal non-tumorigenic diploid cells, expressing 

gap-junctional protein connexin43 and communicating via gap-junctional channels, 

which may differentiate into functional hepatocytes or cardiomyocytes upon injection 

into a syngenic Fischer 344 rat, WB-ras cells are highly tumorigenic in vivo and show 


aberrant expression, localization and phosphorylation of connexin43 in vitro. They are 

also characterized by a loss of contact inhibition of growth and capability of anchorageindependent 

growth. 

Chemopreventive and anticancer effects of phytochemicals tested were evaluated on 

three different levels (endpoints). Restoration of oncogene-inhibited gap junctional 

intercellular communication (GJIC), which represents a key cellular event linked to 

tumor promotion and chemoprevention, was investigated in WB-ras cell line using 

scrape loading/dye transfer assay. Selective antiproliferative and/or cytotoxic effects of 

phytochemicals towards tumorigenic WB-ras cells were assessed by neutral red uptake 

assay and compared with phytochemical effects in normal non-tumorigenic WB-F344 

cell line. In addition to that, alterations of cell cycle induced by phytochemial treatment 

were also studied in both cell lines using propidium iodide staining and flow cytometry 

measurement. 

The results of conducted experiments suggested that tested phytochemicals were able 

to selectively affect evaluated parameters in tumorigenic WB-ras cells, indicating their 

chemopreventive and anticancer activity as well as possible alterations of ras-dependent 

signaling. The employed in vitro models/methods were thus demonstrated to be a 

valuable and effective tool for future phytochemical and dietary compound screening 

for cancer chemopreventive and anticancer activity and for further characterization of 

their mechanisms of action. 


This work was supported by a research grant of the Ministry of Education, Youth and Sports 

LH12034 (CHEMOPREV) - Novel in vitro approach for identification of chemopreventive 

effects and mechanisms of phytochemicals (LH KONTAKT II funding programme). 

P20. IMMUNOMODULATORY PROPERTIES OF CANDIDA GLABRATA CELL WALL 

MANNAN 

Lucia Paulovičová 1 , Ema Paulovičová 1 , Eva Pericolini 2 , Elena Gabrielli 2 , Anna Vecchiarelli 2 

1 

Center of Excellence Glycomed, Department of Immunochemistry of Glycoconjugates, 

Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic; 

chemluli@savba.sk 

2 

Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, 

University of Perugia, Perugia, Italy 

The yeasts Candida glabrata is a human commensal fungus that resides on the skin, 

mucosa surfaces and gastrointestinal tract of healthy individuals. Although C. glabrata is 

not pathogenic under normal host conditions, it can cause infections when host defence 

is compromised. In Slovakia C. glabrata was the second abundant human pathogen 

according to the frequencies in clinical isolates from upper (16.3%), low respiratory 

tract (24.5%) and gynaecological swabs (6%) following C. albicans (Mycology Labs., 

HPL ,Slovakia). This fungus is associated with a wide spectrum of diseases in humans, 

ranging from acute superficial infections of skin and mucosal membranes due to impaired 


epithelial barrier functions in immunocompetent individuals up to inflammatory diseases 

and severe life-threatening infections in immunocompromised patients. C. glabrata is of 

special importance because of a recent increase in its frequency and its innately reduced 

susceptibility to antifungal agents, specifically to azoles. The immunomodulation of 

fungal infections depends on the specific recognition of cell-wall antigens, representing 

the pathogen associated molecular patterns by immunocompetent cells. These antigens 

could be able to induce proliferation and activation of immune cells. The outermost layer 

of the cell wall of Candida species consists of mannoproteins containing O-glycosylated 

oligosaccharide and N-glycosylated polysaccharide moieties. Both carbohydrate 

moieties have been shown to play an essential role in host-fungal interactions, including 

the triggering and modulation of the anti-Candida host immune responses, which 

appear to rely on the interplay between the innate and adaptive immunities. Until 

yet, the role of mannan on immune system modulation is not sufficiently described. 

The dendritic cells (DCs) model represents a promising target for immunotherapy 

interventions and vaccine development. DCs increase an innate or specific response to 

fungi by producing inflammatory mediators, they are uniquely appropriate for decoding 

the fungus-associated information and next translation into a qualitatively different T 

helper responses. We observed, that C. glabrata mannan is able to induce activation 

of DCs and to increase the expression of co-stimulatory molecules CD80 and CD86. C. 

glabrata mannan was also able to stimulate proliferation of mouse splenocytes and 

increase production of TNF-α and IL-4 in concentration dependent manner. 


This contribution is the result of project implementation: Centre of Excellence for 

Glycomics, ITMS 26240120031, supported by the Research & Development Operational 

Programme funded by ERDF. 

This work was supported by the Grant Agency of Slovak Academy of Sciences VEGA 

2/0026/13 and by FEMS Research Fellowship FRF 2010-2. 


Fig.1: Expression of CD80 and CD86 

DCs stimulated for 24 h with 10 μg/ml E. coli lipopolysaccharide (LPS10), 100μg/ml 

mannan C. glabrata (M100), 300μg/ml mannan C. glabrata (M300) and 600μg/ml 

mannan C. glabrata (M600). 


P21. ASSESSMENT OF MITOCHONDRIAL FUNCTIONS 

Barbora Pavlů, Jan Černý 

Faculty of Science, Charles University in Prague, Czech Republic; 

barbora.pavlu@natur.cuni.cz 

Mitochondria have become targets for activating specific killing of cancer cells and as 

a consequence this creates a need for assessment of drugs targeting mitochondria. 

To test effects of these substances - so called ”mitocans”( Ralph et al., 2006) - on 

mitochondria we employed MitoTracker ® Red CMXRos ( Molecular Probes® Invitrogen 

detection technologies), that is chloromethyl-X-rosamine. This fluorescent probe is 

retained in active mitochondria and its accumulation is dependent upon membrane 

potential. Results obtained with this mitochondrion selective probe by FACS analysis 

correspond with qualitative data from fluorescence microscopy. We found that working 

concentration for FACS analysis is between 2-5 nM. 

Suggested mechanism of induction of apoptosis by mitocans is a production of reactive 

oxygen species (ROS), which affect mitochondria in multiple ways (Neužil et al., 2006). 

To test production of ROS in cells treated by mitocans we used CellROX ® Green Reagent 

( Molecular Probes® Invitrogen detection technologies). This probe is non-fluorescent in 

reduced state. After oxidation it becomes fluorescent and binds to DNA. Once again data 

from FACS analysis were in agreement with fluorescence microscopy. We found that 

working concentration for FACS analysis is between 10-30 µM. 

These two probes are compatible for use in multicolour experiments and are suitable for 

assessment of mitochondrial functions in cells treated by mitocans. 


This work was supported by Grant Agency of the Czech Republic (P302/13/16565S), the 

Czech Ministry of Education, Youth and Sports of the Czech Republic (MSM0021620858) 

and by the grant SVV (265211). 

References 

Neuzil J, Wang XF, Dong LF, Low P, Ralph SJ.: Molecular mechanism of ‘mitocan’-induced 

apoptosis in cancer cells epitomizes the multiple roles of reactive oxygen species and 

Bcl-2 family proteins. - FEBS Lett. 2006 Oct 2;580(22):5125-9. 

Ralph SJ, Low P, Dong L, Lawen A, Neuzil J.: Mitocans: mitochondrial targeted anti-cancer 

drugs as improved therapies and related patent documents. - Recent Pat Anticancer 

Drug Discov. 2006 Nov;1(3):327-46. 


P22. HUMAN INTERLEUKIN-23 RECEPTOR ANTAGONISTS DERIVED FROM AN 

ALBUMIN-BINDING DOMAIN SCAFFOLD INHIBIT IL-23-DEPENDENT EX VIVO 

EXPANSION OF IL-17-PRODUCING T-CELLS 

Ondřej Pelák 1 , Milan Kuchař 2 , Lucie Vaňková 2 , Hana Petroková 2 , Jiří Černý 3 , Radim 

Osička 4 , Hana Šípová 5 , Bohdan Schneider 3 , Jiří Homola 5 , Peter Šebo 2,4 , Tomáš Kalina 1 

and Petr Malý 2 

1 

Department of Pediatric Hematology and Oncology, 2 nd Faculty of Medicine, Charles 

University and University Hospital Motol, Prague, Czech Republic 

2 

Laboratory of Ligand Engineering and 3 Laboratory of Molecular Recognition, Institute 

of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech Republic 

4 

Institute of Microbiology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech Republic 

5 

Institute of Photonics and Electronics AS CR, v. v. i., Chaberská 57, 182 51, Prague, 


pelak.ondrej@gmail.com 

Autoimmune diseases such as psoriasis, Crohn’s disease, rheumatoid arthritis or multiple 

sclerosis, have recently been found to be associated with IL-23-mediated signaling and 

increased TH17 lymphocytes. Thus, inhibition of IL-23 might provide therapeutic effect. 

Engineered recombinant binders from small protein scaffolds of the albumin binding 

domain of streptococcal protein G can be generated with binding affinities similar to 

monoclonal antibodies. These binders can exert inhibitory function by blocking of the 

cytokine binding epitope on its receptor. 

We tested the inhibition effect of recombinant binders of human interleukin-23 receptor 

(IL-23R) on primary human CD4+ T-cells. Peripheral mononuclear cells (PBMNC) were 

isolated from blood of healthy donors and stimulated with anti-CD3 antibody and 

co-stimulated by activating antibodies CD28 and CD49d in presence of the TH-17 

conditioning cytokines Il-23 and Il-2 for 3 days. After 3 days the intracellular cytokine 

staining protocol was used to detect production of IL-17 and INF-γ to see the inhibition 

effect of novel IL-23R-binding proteins. We show that presence of REX009, REX115 and 

REX125 variants of the IL-23R-binding proteins inhibited the IL-23-driven expansion of 

IL-17- producing primary human CD4+ T-cells even by 75%. 

To confirm direct inhibitory effect of REX binders on T-cells, we repeated the same 

experiment in samples with separated T-cells with similar results. 

In summary, we show that REX variants inhibited IL-23-dependent TH-17+ cell expansion 

to the level of samples with no IL-23 added. While IL-23 was shown to promote the 

development of TH-17+ T-cells in man, the mode of this action might be fixing the TH- 

17 commitment rather than “de novo” TH-17 development, survival or proliferation 

enhancement (Stritesky et al, 2008). 

References 

Stritesky GL, Yeh N, Kaplan MH. IL-23 promotes maintenance but not commitment to the 

Th17 lineage. J Immunol 2008;181(9):5948-5955. 


OP and TK are supported by MH CR, DRO 00064203 


P23. PTEROSTILBENE: COMPARISON OF ANTI-INFLAMMATORY EFFECTS IN VITRO AND 

IN VIVO 

Tomas Perecko 1,2 , Gabriela Ambrozova 2 , Antonin Lojek 2 , Milan Ciz 2 , Radomir Nosal 1 , 

Juraj Harmatha 3 , Katarina Drabikova 1 , Viera Jancinova 1 

1 

Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, 

Dubravska cesta 9, 841 04 Bratislava, Slovak Republic 

2 

Institute of Biophysics, Academy of Sciences of the Czech Republic, v. v. i., Kralovopolska 

135, 612 65 Brno, Czech Republic, tomas.perecko@ibp.cz 

3 

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech 

Republic v.v.i., Flemingovo namesti 2, 166 10 Prague, Czech Republic 

Neutrophils and macrophages (professional phagocytes) are a crucial part of the innate 

immune system. In response to infection or foreign particles, they produce reactive 

oxygen and nitrogen species (ROS/RNS) to destroy the invading pathogens. The dark 

side of phagocyte activity is their contribution to tissue damage. This is due to overproduction 

of ROS/RNS seen in many inflammatory diseases, e.g. rheumatoid arthritis 

(Witko-Sarsat et al., 2000). 

Rheumatoid arthritis is a chronic autoimmune inflammatory disease characterised by 

bone erosion and cartilage damage with synovial hyperplasia. Primed neutrophils and 

macrophages are present in the synovial fluid and on the panus-cartilage interface in 

arthritis. Thus, activated phagocytes by producing ROS/RNS could contribute to joint 

destruction (Edwards and Hallett, 1997). This highlights the importance of searching for 

therapeutic agents capable to control the production of ROS/RNS in chronic inflammatory 

diseases. 

In this study, the effects of pterostilbene (stilbene type polyphenol) on neutrophil 

ROS production and macrophage RNS generation were investigated in isolated human 

neutrophils and murine macrophage RAW264.7 cell line in vitro. Since apoptosis 

of neutrophils and their subsequent phagocytosis by macrophages is important for 

resolution of inflammation (Fox et al., 2010), the effect of pterostilbene on neutrophil 

apoptosis was investigated by using annexin-V and propidium iodide staining. Lewis 

rat arthritis model was used for evaluation of pterostilbene effects on inflammation in 

vivo. Solvent agent was administered to the healthy control group and arthritic control 

group of animals. In the third group, arthritic animals received pterostilbene 30 mg/kg, 

daily, p.o. The number and activity of neutrophils in blood were measured at weekly 

basis during the whole experiment (21 days). Moreover, the GM-CSF in plasma of tested 

animals was analysed by using cytometric bead assay. 

Pterostilbene decreased the production of ROS and RNS in vitro. However, the 

mechanisms differ: pterostilbene scavenged ROS and down-regulated the iNOS 

expression without interacting with NO. There was no effect on neutrophil apoptosis in 

vitro. In the pterostilbene treated arthritic group, the treatment significantly lowered the 

number of neutrophils in blood on day 14 and 21 without significant down-regulation 

of neutrophil oxidative burst. Pterostilbene had no effect on GM-CSF level in arthritic 

animals. 


These results indicate that the promising in vitro effects of pterostilbene on ROS/RNS 

production operate by different mechanisms and its beneficial activity in experimental 

arthritis may be attributed to regulation of neutrophil numbers. 


Supported by the grants APVV-0052-10 and 0315-07, VEGA-2/0010/13 and 

CZ.1.07/2.3.00/30.0030. 

References 

Edwards, S. W., and Hallett, M. B.: Seeing the wood for the trees: the forgotten role of 

neutrophils in rheumatoid arthritis. - Immunology Today 18: 320-324, 1997. 

Fox, S., Leitch, A. E., Duffin, R., Haslett, C., Rossi, A. G.: Neutrophil apoptosis: relevance 

to the innate immune response and inflammatory disease. – Journal of Innate Immunity 

2: 216-227, 2010. 

Witko-Sarsat, V., Rieu, P., Descamps-Latscha, B., Lesavre, P., Halbwachs-Mecarelli, 

L. Neutrophils: molecules, functions and pathophysiological aspects. - Laboratory 

Investigation 80: 617-653, 2000. 

P24. Β-ARRESTIN PROMOTES WNT-INDUCED LRP6 PHOSPHORYLATION VIA 

INCREASED MEMBRANE RECRUITMENT OF AMER1 

Vítězslav Kříž 1,2* , Vendula Pospíchalová 1*# , Jan Mašek 1,6 , Michaela Brita Christina 

Kilander 4 , Josef Slavík 5 , Kristina Tanneberger 3 , Gunnar Schulte 1,4 , Miroslav Machala 5 , 

Alois Kozubík 1,2 , Juergen Behrens 3 and Vítězslav Bryja 1,2 

1 

Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech 

Republic 

2 

Department of Cytokinetics, Institute of Biophysics, Academy of Science of the Czech 

Republic, Brno, Czech Republic 

3 

Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany 

4 

Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden 

5 

Department of Toxicology, Pharmacology and Immunotherapy, Veterinary Research 

Institute, Brno, Czech Republic 

6 

Institute of Molecular Genetics, Academy of Science of the Czech Republic, Prague, 


*equal contribution 

# 

contact email: pospich@sci.muni.cz 

β-arrestin is a scaffold protein, which regulates signal transduction by seven 

transmembrane spanning receptors. Among other functions it is also critically required 

for Wnt/β-catenin signal transduction (for review see Schulte G, et al., 2010). In the 

present study we provide for the first time a mechanistic basis for the β-arrestin function 

in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient 

Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling (Pan W, 

et al., 2008). β-arrestin regulates Lrp6 phosphorylation via a novel interaction with 

phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P 2 

)-binding protein Amer1/WTX/ 


Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled 

(Dvl)-associated PtdIns(4,5)P 2 

production to the phosphorylation of Lrp6 (Tanenberger 

K, et al., 2011). We show here that β-arrestin is required for the Wnt3a-induced Amer1 

membrane dynamics (Fig.1) and downstream signaling. Finally we show that β-arrestin 

interaction with PtdIns kinases is responsible for Wnt3a-induced PtdIns(4,5)P 2 

formation 

(PI4KIIα and PIP5KIβ). Importantly, cells lacking β-arrestin showed higher steady state 

levels of relevant PtdInsP and were unable to increase levels of these PtdInsP in response 

to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 

phosphorylation by the regulation of the membrane dynamics of Amer1. We propose 

that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5) 

P 2 

, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dvl-associated 

kinases. 

Figure 1. Wnt3a is unable to regulate AMER1 dynamics in the absence of β-arrestin 

and in the case of AMER1(2-838aa) construct, which lacks the β-arrestin interaction 

domain. 


This work was supported by Czech Science Foundation (204/09/0498, 204/09/J030), 

EMBO Installation Grant, the Ministry of Education Youth and Sport of the Czech 

Republic (MSM0021622430). GS is supported by Knut and Alice Wallenberg Foundation 

(KAW2008.0149), Swedish Research Council (K2008-68P-20810-01-4, K2008- 

68K-20805-01-4) and The Swedish Foundation for International Cooperation in Research 

and Higher Education (STINT). JB is supported by grant Be1550/6-1 from Deutsche 

Forschungsgemeinschaft (DFG). The collaboration between Masaryk University and 

Karolinska Institutet is supported by by the European Regional Development Fund (KI- 

MU; CZ.1.07/2.3.00/20.0180). 

References 

1. Pan, W., S. C. Choi, H. Wang, Y. Qin, L. Volpicelli-Daley, L. Swan, L. Lucast, C. Khoo, 

X. Zhang, L. Li, C. S. Abrams, S. Y. Sokol, and D. Wu. 2008. Wnt3a-mediated formation 

of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation. Science 

321:1350-1353. 

2. Schulte, G., A. Schambony, and V. Bryja. 2010. beta-Arrestins - scaffolds and signalling 

elements essential for WNT/Frizzled signalling pathways? Br J Pharmacol 159:1051-1058. 

3. Tanneberger, K., A. S. Pfister, K. Brauburger, J. Schneikert, M. V. Hadjihannas, V. Kriz, G. 

Schulte, V. Bryja, and J. Behrens. 2011. Amer1/WTX couples Wnt-induced formation of 

PtdIns(4,5)P2 to LRP6 phosphorylation. Embo J 30:1433-1443. 


Fig. 1: Analysis of AMER1 membrane dynamics in presence and absence of β-arrestin 


in MEF cells reveals requirement of the interaction between AMER1 and β-arrestin for 

Wnt3a-induced AMER1 dynamics. (A-C) WT and β-arr1/2 DKO MEFs were transfected 

with EGFP-AMER1 (A, B) or EGFP-AMER1(2-838) (C) and membrane dynamics of the 

constructs was analyzed by Fluorescence Recovery After Photobleaching (FRAP) method. 

Example of cells subjected to FRAP is given on the left, squares indicate bleached regions. 

On the right statistical analysis of FRAP experiment using WT and β-arr1/2 DKO MEFs 

stimulated with PBS or Wnt3a is shown. The graphs show mean values + s.e.m., and 

the best fitting curve model, which was used for calculation of mobile pool of EGFP- 

AMER1 (% of fluorescence recovered) and of the recovery halftime (T1/2). N – number 

of analyzed cells. 

P25. DETECTION OF CANCER STEM CELL MARKERS AND ANALYSIS OF EPITHELIAL-TO- 

MESENCHYMAL TRANSITION IN PROSTATE BENIGN AND CANCER CELL LINES 

Eva Sedlmaierová 1,2* , Zuzana Pernicová 1,3 , Šárka Šimečková 1,2 , Eva Slabáková 1,3 , Radek 

Fedr 1 , Alois Kozubík 1,2 , Karel Souček 1,3* 

1 


Republic, Brno, Czech Republic; 

2 

Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, 

Czech Republic; 

3 


St. Anne´s University Hospital Brno, Brno, Czech Republic 

*Correspondence to: sedlmaierova@ibp.cz, ksoucek@ibp.cz 

Cancer stem cells (CSCs) have long been implicated in numerous tumour formations 

and therapy resistance including prostate cancer disease. Importantly, mechanisms of 

their origin have not been elucidated in prostate cancer yet. It has been demonstrated 

that epithelial-to-mesenchymal transition (EMT) is phenomenon which can lead to the 

acquisition of stem cell properties in various types of cancer. This was first discovered 

by research on immortalized human mammary epithelial cells, where induction of EMT 

resulted in the acquisition of mesenchymal properties and in the expression of stem 

cell markers. Furthermore those cells had properties similar to mammary epithelial 

stem cells (Mani et al., 2008). In prostate cancer, cells with EMT phenotype have been 

shown to express stemness factors and to have increased clonogenic capacity (Kong et 

al., 2010). However phenotypic heterogeneity and EMT status of routinely cultivated 

prostate cell lines remains unknown. 

In this study we aimed to screen surface expression of selected CSCs markers (Trop2, 

CD133, CD44, CD24 and CD49f) in a panel of prostate benign and cancer cell lines using 

multicolour flow cytometry. We found out that each cell line express different set and 

combinations of these CSCs markers. Next, to elucidate the EMT status in putative 

CSCs subpopulations, we combined analysis of selected surface stem cell markers with 

detection of regulators and markers of EMT (Snail, Slug, E-cadherin, N-cadherin) in 

CD24/CD44 subpopulation of PC-3 and DU-145 cells. 


In summary, we described different subpopulations of CSCs in a panel of prostate 

epithelial cell lines and determined EMT status of those subpopulations. 



and by project FNUSA-ICRC (no. CZ.1.05/1.1.00/02.0123) from the European Regional 



References 

Kong, D., S. Banerjee, et al. (2010). “Epithelial to Mesenchymal Transition Is Mechanistically 

Linked with Stem Cell Signatures in Prostate Cancer Cells.” PLoS ONE 5(8): e12445. 

Mani, S. A., W. Guo, et al. (2008). “The Epithelial-Mesenchymal Transition Generates 

Cells with Properties of Stem Cells.” Cell 133(4): 704-715. 

P26. POTENTIAL INHIBITORS OF GLUCOSYLCERAMIDE SYNTHASE AND THEIR EFFECT 

ON CELL VIABILITY AND CALCIUM TRANSPORT 

Boris Lakatoš* 1 , Katarína Turáková 1 , Daniela Moravčíková 2 , Andrej Duriš 2 , Dušan Berkeš 2 

1 

Department of Biochemistry and Microbiology, Faculty of Chemical and Food 

Technology, Slovak University of Technology, Bratislava, Slovak Republic; 

2 

Department of Organic Chemistry, Faculty of Chemical and Food Technology, Slovak 

University of Technology, Bratislava, Slovak Republic; 

*boris.lakatos@stuba.sk 

Glucosylceramide (GlcCer) is a fundamental glycosylated lipid found in organisms ranging 

from mammals to fungi. It is composed of a hydrophilic β-linked glucose and a hydrophobic 

ceramide. Mammalian GlcCer mainly contains sphingosine (d18:1), a sphingoid base 

that has one double bond at the C4 position in a trans conformation, and N linked C16– 

C24 fatty acids. GlcCer is the precursor of hundreds of different glycosphingolipids. This 

cerebroside is synthesized from uridine diphosphate-glucose and ceramide by a GlcCer 

synthase (UDP-glucose:ceramide glucosyltransferase; UGCG, GlcT-1, CGT, or GCS, EC 

2.4.1.80). GlcCer-based sphingolipids have been identified as important mediators of a 

variety of cellular functions, including proliferation, differentiation, development, and 

cell-cell recognition (1). There is several pathological situations which are related with 

disequilibrium of sphingolipids in cells, e.g. cancer, diabetes, neurological diseases (2). 

Aim of this work was to study effects of several newly synthesized derivatives of (±)-threo- 

1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which is known inhibitor 

of GlcCer synthase, on activity of this enzyme, cell viability and calcium transport. As 

a model cells we used thymocytes isolated from thymus of 3-7 weeks old mice (ICR 

strain). Changes of calcium transport were measured using Ca 2+ sensitive probe Fluo-3 

on spectrofluorometer FluoroMax-4. The activity of GlcCer synthase was assessed from 

lipid extracts of cells cultivated with derivatives by thin layer chromatography using NBD- 

C12-ceramide. HPTLC plates were analyzed using phosphoimager Typhoon 9210 and cell 

viability was determined by flow cytometry and direct cell counting. 


Effects of studied newly synthesized PDMP derivatives were dependent on their chemical 

structure and duration of cell cultivation. Several derivatives were able to change 

Ca 2+ transport already after 15 minutes of cultivation, but their effect of cell viability 

manifested only after 12 h of cultivation. Activity of GlcCer synthase was affected in case 

of 4 from 12 studied compounds after longer cultivation, but was not in relation with 

changes in calcium transport and cell viability. 


This work was supported by grant agency VEGA No. 01/0471/11 and 01/0441/11 

References: 

1. Hakomori, S. and Igarashi, Y. (1993) Adv. Lipid Res. 23, 147–162 

2. Lahiri, S. and Futerman, A. H. (2007) Cell. Mol. Life Sci. 64 (2007) 2270 – 2284 

P27. DOCOSAHEXAENOIC ACID EFFECTIVELY STIMULATES TRAIL-INDUCED APOPTOSIS 

OF COLON CANCER CELLS AT THE LEVEL OF MITOCHONDRIA 

Belma Skender 1, 2 , Björn Kruspig 3 , Vladimir Gogvadze 3 , Jiřina Hofmanová 1, 2 , Alois 

Kozubík 1, 2 , Boris Zhivotovsky 3 , Alena Hyršlová Vaculová 1 

1 


Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic; 

2 

Department of Animal Physiology and Immunology, Faculty of Science, Masaryk 

University, Terezy Novákové 64, 621 00 Brno, Czech Republic 

3 

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 

210, Stockholm, SE-171 77 Sweden 

belma@ibp.cz, vaculova@ibp.cz 

Docosahexaenoic acid (DHA), an essential n-3 polyunsaturated fatty acid, is known for 

its beneficial effect in a wide variety of diseases ranging from autoimmune disorders 

to several types of malignancies including colorectal cancer. During carcinogenesis, 

mitochondria-dependent apoptotic pathways are very frequently suppressed, which 

results in attenuation of cell death and enhanced proliferation. Mitochondria belong 

to the subcellular sites where n-3 fatty acids are rapidly incorporated. DHA containing 

six double bonds causes cardiolipin structural changes and increases mitochondrial 

membrane fatty acids unsaturation, their susceptibility to oxidative stress and alters 

membrane barrier properties. We hypothesize that DHA might facilitate the toxic effects 

of certain antitumour agents, which could directly and specifically reinforce apoptosis in 

cancer cells. Our aim was to elucidate a potential sensitizing effect of DHA on apoptosis 

induced by a cytokine of the tumor necrosis factor (TNF) family - TRAIL (TNF-related 

apoptosis inducing ligand) in colon cancer cells. TRAIL has been shown to selectively 

induce apoptosis in tumor, but not in normal cells. However, many tumor cells may still 

exhibit a TRAIL-resistant phenotype, which is currently a major obstacle in TRAIL-based 

therapy. 

Here, we demonstrate that DHA is capable to increase significantly apoptosis induced 

by this cytokine and highlight the importance of the mitochondrial apoptotic pathway in 


this process. Combined treatment with TRAIL and DHA markedly stimulated apoptosis 

in SW620 cells, which included the activation of proapoptotic Bcl-2 family proteins Bak 

and Bax, facilitated permeabilization of the outer mitochondrial membrane and release 

of cytochrome c, decrease in mitochondrial membrane potential and inhibition of 

mitochondrial respiration. 

In summary, our results show that DHA can be used to enhance TRAIL-induced apoptosis 

in resistant colon cancer cells by targeting mitochondria. 

This work was supported by grants Nos. P301/11/1730, 13-097665 of the Czech Science 

Foundation and MSM0021622430 Ministry of Education, Youth and Sports. 

P28. ANALYSIS OF MIGRATION AND INVASION PROPERTIES ASSOCIATED WITH 

CANCER TRANSFORMATION OF HUMAN EPITHELIAL CELLS 

Eva Slabáková 1,2 , Veronika Toporcerová 1,2,3 , Radek Fedr 1 , Antonín Hlaváček 4 , Petr 

Skládal 4 , Alois Kozubík 1,3 , Karel Souček 1,2 

1 

Academy of Science of the Czech Republic, Institute of Biophysics, Department of 

Cytokinetics, Královopolská 135, 61265 Brno, Czech Republic 

2 

International Clinical Research Center, St. Anne‘s University Hospital Brno, Brno, Czech 

Republic 

3 

Department of Experimental Biology, Masaryk University, Faculty of Science, Brno, 


4 

Centre for Structural Biology, Central-European Technology Institute, Brno, Czech 

Republic. 

Correspondence to: slabakova@ibp.cz 

Most cancer–related deaths are associated with advanced disease and metastasis. 

Epithelial-mesenchymal transition (EMT) facilitates cancer cell dissemination and 

metastasis by changes in cell polarity, cell-cell adhesions and organization of cytoskeleton. 

Moreover, EMT can also facilitate survival of metastasizing cells by endowing them with 

cancer stem cell properties. 

Cell adhesion, interaction with extracellular matrix (ECM) and reorganization of 

cytoskeleton are crucial processes implicated in cell migration and invasion. While 

certain cell types invade through the ECM using extracellular protein degradation by 

enzymatic activity of matrix metalloproteases (MMP), other cell types rely on their 

ability to reorganize the cytoskeleton and are characterized by an amoeboid mode of 

migration which does not involve ECM degradation. 

Analyzing paired benign and tumorigenic cell lines derived from benign human prostate 

or breast tissue, we observed that cancer transformation is often accompanied by EMT, 

as evidenced by decreased expression of epithelial markers and increased expression of 

mesenchymal markers and EMT regulating transcription factors. To confirm that these 

changes in gene and protein expression and localization are functionally implicated in cell 

motility, we compared the migration and invasion potential of benign and transformed 


cell lines using multiple methodical approaches. Human metastatic cancer cell lines 

were used as positive controls. 

The goal of this work was to find an optimal functional assay which could be used for 

subsequent functional assessment of specific molecules in cell migration and invasion. 

To study cell migration, we performed chemotactic migration assays using transwell 

chambers or real-time analysis with xCELLigence system. Alternatively, a “wound healing 

- scratch assay” or agarose spot invasion assay was tested. Cell invasion was studied in a 

similar setup to cell migration, after coating the membranes with ECM proteins such as 

collagen or Matrigel. Activity of ECM degrading enzymes was analyzed by zymography 

or a fluorescence microscopy-based gelatinase assay, expression of MMP proteins was 

evaluated by western blotting. 

Cell migration towards chemoattractant was enhanced by starving the cells in growth 

factor depleted media. We observed a specific and robust activation of TGF-beta 

signaling pathway under these conditions. These results were correlated with expression 

of a set of secreted proteins on a cytokine array. Altogether, we observed that cancer 

transformation of human benign prostate hyperplasia-derived BPH-1 cells is associated 

with increased migration potential, but not with significant enhancement of cell invasion 

associated with ECM protein degradation. 

This work was supported by grant no. P301/12/P407 of the Czech Science Foundation; 

by grant no. NT13573-4/2012 of the Ministry of Health of the Czech Republic; by the 

Academy of Sciences of the Czech Republic, grant no. AV0Z50040702, and by the project 

FNUSA-ICRC no. CZ.1.05/1.1.00/02.0123 from the European Regional Development 

Fund. 

References: 

Slabakova, E., et al., TGF-beta1-induced EMT of non-transformed prostate hyperplasia 

cells is characterized by early induction of SNAI2/Slug. Prostate, 2011. 

P29. NKD1- CREER T2 MOUSE STRAIN: A NEW TOOL FOR SITE-SPECIFIC 

RECOMBINATION IN WNT RESPONSIVE CELLS OF MOUSE INTESTINE AND LIVER 

Fafílek Bohumil 1 , Stančíková Jitka 1,2 , Hlavatá Adéla 1,2 , Kořínek Vladimír 1 

1 

Laboratory of Cell and Developmental Biology, IMG AV CR (jitka.stancikova@img.cas.cz) 

2 

Faculty of Science, Charles University in Prague 

Wnt signaling pathway plays a crucial role in ontogenesis and development of 

all metazoans. In adult mammals, the Wnt signaling pathway is required for the 

maintenance of the intestinal homeostasis and establishment of proper hepatic zonation. 

In contrary to that, aberrant activation of the Wnt pathway leads to neoplasia and cancer 

development, notably in the intestine and liver. 

To investigate the role of the Wnt pathway in gut epithelium homeostasis and its 

malignant transformation we employed chromatin immunoprecipitation method (ChIP) 

in combination with DNA microarrays (so-called ChIP-on-chip) to identify genes regulated 

by the Wnt signaling. One of the most prominent targets was the NKD1 (Naked Cuticle 


Homolog 1) gene; previously identified as a Wnt-induced intracellular negative regulator 

of the canonical Wnt signaling. 

With use of BAC recombineering, we generated mice with CreER T2 recombinase produced 

corresponding to the gene NKD1 (NKD1-CreER T2 ) expression. Comparing the natural 

NKD1 expression with transgenic Cre in NKD1-Cre x Rosa-lacZ reporter strain hybrids 

proved that the transgenic mouse produces Cre in NKD1 + cells only. Two of the most 

interesting sites of the NKD1-CreER T2 expression in adult mice are perivenous hepatocytes 

and intestinal crypt compartment, which was confirmed by the expression profiling. New 

mouse strain NKD1-Cre ER T2 is therefore a unique tool for gene manipulation particularly 

in hepatocytes localized in the perivenous zone. 

P30. EFFECTS OF CHOLESTANE BRASSINOSTEROID DERIVATIVES ON HORMONE-IN/ 

SENSITIVE BREAST AND PROSTATE CANCER CELL LINES 

Steigerová J. 1,2 , Rárová L. 3 , Křížová K. 2 , Oklešťková J. 4 , Šváchová M. 5 , Levková M. 2 , Kolář 

Z. 1,2 a Strnad M. 3,4 

1 

Laboratory of Molecular Pathology, Department of Clinical and Molecular Pathology, 

Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15 Olomouc, 


2 

Institute of Molecular and Translation Medicine, Faculty of Medicine and Dentistry, 

Palacký University and Faculty Hospital in Olomouc, Puškinova 6, 775 20 Olomouc, 


3 

Centre of the Region Haná for Biotechnological and Agricultural Research, Department 

of Growth Regulators, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 

Olomouc, Czech Republic 

4 

Laboratory of Growth Regulators, Palacký University & Institute of Experimental 

Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic 

5 


Palacký University and University Hospital Olomouc, I. P. Pavlova 6, 775 20 Olomouc, 


Brassinosteroids (BRs), polyhydroxylated sterol derivatives with close structural similarity 

to animal and insect steroid hormones, are plant growth regulators representing a 

group of newly-discovered agents with relatively wide-ranging effects in plants. Based 

on a structural domain similarity between steroids and BRs, the brassinosteroid 

cytotoxic activity could be, at least partially, related to brassinosteroid-steroid receptor 

interactions. Investigation of the mechanisms of action of BRs in human cancer cells 

using cellular and molecular techniques indicated the possible involvement of steroid 

receptors in BR action. Understanding the mechanisms of nuclear receptor action 

will enhance our knowledge of transcription and hormone influences on disease and 

facilitate the design of drugs with greater therapeutic value. 

Molecular and cellular effects of natural BRs and their synthetic cholestane derivatives 

were examined in human hormone-in/sensitive breast (MCF-7, MDA-MB-68) and 


prostate (LNCaP, DU-145) cancer cell lines. The effects of treatments with studied agents 

on cancer cells were surveyed using flow cytometry, reporter assay, Western blotting, 

TUNEL assay and immunofluorescence analyses. 

We found that these agents inhibited cell proliferation and induced G 1 

-phase cell cycle 

arrest in association with alterations of cell cycle related protein expression. In hormonedependent 

cell lines, treatment with studied compounds resulted in changes in the 

expression and subcellular distribution of nuclear steroid hormone receptors (ER-α, ER-β 

and AR). KAR6299 was by far the most potent compound from the whole set of tested 

cholestane derivatives and it activated ER-α, ER-β and AR. It showed a slight selectivity 

toward ER-α compared to ER-β. Changes in ER-α and ER-β localization patterns were 

observed in MCF-7 cells after 24 h treatment. KAR6299 downregulates ER-α in MCF-7 

cells through an unknown mechanism which probably compensates for its estrogenic 

activities in long term proliferation studies and results in inhibition of proliferation of 

both hormone-sensitive and hormone-insensitive cell lines. 

The mechanisms of action of BRs in animal cells are still largely unknown, but it seems 

possible that they may interact with one or more of the numerous steroid-binding 

proteins. Apparently there are multiple effects, both steroid receptor-dependent and 

-independent. Thus comparison of the effects of natural BRs and their analogues on 

receptors, and detailed characterization of their biological effects, could both provide 

extend fundamental knowledge of hormone-receptor interactions and could have 

valuable practical applications. 


This work was supported by grant from the Ministry of Health of the Czech Republic 

(NT11060), grant ED0007/01/01 of Centre of the Region Haná for Biotechnological 

and Agricultural Research, grants No. IAA400550801 and 1M06030. Infrastructural 

part of this project (Institute of Molecular and Translational Medicine) was supported 

from the Operational Programme Research and Development for Innovations (project 

CZ.1.05/2.1.00/01.0030). 

P31. COMBINED TREATMENT OF PPAR LIGAND WITH OXALIPLATIN AFFECT THE 

CELL CYCLE AND DEATH IN COLON CELL LINE BOTH SENSITIVE AND RESISTANT TO 

OXALIPLATIN 

Strakova N. 1,3 , Hofmanova J. 1 , Zapletal O. 1 , Soucek K. 1,2 , Fedr R. 1 , Bouchal J. 3 , 

Ehrmann J. 3 , Kolar Z. 3 , Hyrslova Vaculova A. 1 , Tylichova Z. 1 , Laukova J. 1 , Kozubik A. 1 

1 


Republic, v.v.i., Brno, Czech Republic; strakova@ibp.cz 

2 



3 


Palacky University Olomouc, Olomouc, Czech Republic 

PPAR (Peroxisome Proliferator - Activated Receptors) are transcription factors which 


egulate expression of different genes. PPAR isoforms (PPAR alpha, beta/delta, gamma) 

function through very specific mechanisms. PPAR alpha is predominantly expressed in 

liver, heart muscle and brown adipose tissues. It controls fatty acid oxidation and lipid 

metabolism by regulation of target genes, like ApoA1, the major apolipoprotein in highdensity 

lipoprotein. PPAR alpha agonist turns on ApoA1 expression. PPAR alpha also 

controls the expression of lipoprotein lipase, which hydrolyzes triglycerides. PPAR delta 

is the second member of this family. It is ubiquitously expressed and has crucial role 

in fatty acid oxidation. PPAR gamma is expressed mainly in white and brown adipose 

tissues where it functions as central regulator of adipogenesis but it also has important 

roles in glucose control and insulin sensitivity by regulation expression of target genes. 

In addition, PPAR gamma has been described in many different types of normal and 

cancer cells. All PPAR are activated by specific ligands and then they form complex with 

RXR alpha. Co-repressors are released and co-activators are recruited to this complex. 

Finally, they modify expression of different target genes. The ligand-binding domain for 

these receptors is exceptionally large. There are various ligands for the same receptor 

that would have different responses with different side effect profiles. Thiazolidinediones 

(e.g. rosiglitazone, pioglitazone) are selective PPAR gamma ligands, but their effects in 

cancer monotherapy is not very convincing. 

Oxaliplatin is the newest platinum derivative used in standard chemotherapy acting 

primarily through DNA damage. However, serious side-effects and resistance to 

treatment are the main disadvantages of oxaliplatin. Therefore, the new combined 

chemotherapeutic strategies are considered with the aim to suppress these unfavorable 

effects of platinum-based drugs. 

Our study was focused on the combination of oxaliplatin with PPAR gamma ligand 

(rosiglitazone; RGZ) in human colon cancer cell lines. The results confirmed that 

oxaliplatin and RGZ alone inhibit proliferation of human colon adenocarcinoma cell line 

HT-29 in dose - dependent manner. RGZ caused localization of PPAR gamma receptor 

into the nucleus after 0.5 - 1 hour treatment. Expression of PPAR gamma was decreased 

and localization was translocated perinuclearly after 5 – 48 hour RGZ treatment. Using 

PPAR gamma siRNA we confirmed that the decline of PPAR gamma expression is caused 

by protein degradation after longer time of RGZ application. This verified that RGZ 

decreased either protein expression or nuclear localization in longer time interval. RGZ 

alone slightly increased BrdU (5-bromo-2-deoxyuridine) incorporation into the nucleus 

after 24 hours. Combination of RGZ with oxaliplatin promoted antiproliferative effects 

and cell cycle arrest in the G2/M phase. At the same time we observed enhanced 

apoptosis and increased expression of cell cycle related and DNA damage proteins. We 

suggest that increased percentage of active BrdU cells induced by RGZ may support the 

effects of oxaliplatin. 

Because resistance to oxaliplatin is major problem in colorectal cancer treatment we 

established cell line derived from HT-29 that acquired resistance to 10mM oxaliplatin 

(HT-29 res). We confirmed that in those cells RGZ or oxaliplatin alone did not affect cell 

cycle progression. Interestingly, combination of RGZ and oxaliplatin arrested these cells 

in G2/M phase, similarly to sensitive cells treated only by oxaliplatin. In addition, single 

cell sorting of HT-29 res showed that combination of RGZ with oxaliplatin suppress their 

colony formation ability. 


In summary, our results demonstrated that combination of RGZ with oxaliplatin may be 

more effective then single drug treatment in both sensitive and resistant colon cancer 

cells. Thus, this combination seems to be promising in the treatment of colon cancer 

cells resistant to oxaliplatin. 


This work was supported by grant of Internal Grant Agency of Ministry of Health of the 

Czech Republic No. NT 11201-5/2010, grant Czech Science Foundation No. P301/11/1730 

and FNUSA-ICRC European Regional Development Fund No. CZ.1.05/1.1.00/02.0123. 

P32. SYNTHETIC LETHALITY OF CHK1 INHIBITION AND DNA DAMAGE IN NORMAL 

AND TUMOR CELL LINES 

Tereza Suchánková 1 , Ondřej Hylse 2 , Kamil Paruch 2,3 , Zuzana Pernicová 1,3 , Alois Kozubík 1,4 , 

Karel Souček 1,3 

1 

Academy of Sciences of the Czech Republic, Institute of Biophysics, Královopolská 135, 

61265 Brno, Czech Republic. 

2 

Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic. 

3 

International Clinical Research Center, St. Anne‘s University Hospital Brno, Brno, Czech 

Republic 

4 

Department of Experimental Biology, Masaryk University, Faculty of Science, Brno, 

Czech Republic. 

Correspondence to: suchankovatereza@ibp.cz, ksoucek@ibp.cz 

Progress in understanding of the pathways affected by cancer specific mutations lead 

to novel therapeutic approach called the concept of synthetic lethality. Components of 

DNA damage response pathway were identified as suitable targets of synthetic lethal 

interactions with commonly lost tumor suppressor genes such as p53. Loss of tumor 

suppressor gene alone may not change the response to genotoxic stress induced by 

chemotherapy. However, the inhibition of a second pathway involved in a synthetic lethal 

interaction with this lost suppressor can result in a dramatic difference in sensitivity to 

treatment. In this context, the promising druggable targets are kinases regulating the 

cell cycle checkpoints. 

Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to 

DNA damage. CHK1 inhibitors sensitize tumors to a variety of DNA-damaging agents in 

preclinical models and are being evaluated in clinical trials. SCH 900776 was identified as 

a potent and selective CHK1 inhibitor (Guzi et al., 2011). 

In our work, we proved the hypothesis of synthetic lethality using inhibitors of CHK1 in 

combination with pre-treatment by gemcitabine and hydroxyurea in vitro in normal and 

tumor cells. Our results indicate that these inhibitors enable to reduce the concentration 

of chemotherapeutics to obtain the same growth inhibition. The most significant 

sensitization was observed in human prostate cancer cell lines PC3 and DU-145. The 

effect was also achieved in tumorigenic prostate cells BPH-1 CAFTD03, but only transient 

effect has occurred in their benign counterparts. Further, using the same amount of drug 


was harmful neither alone nor in combination to normal cells, which can be a possible 

rationale for future clinical trials. 


This work was supported by MŠMT grant CZ.1.07/2.3.00/30.0030, by MZD grant 

NT13573-4/2012; by the AVCR grant AV0Z50040702, and by the project FNUSA-ICRC 

CZ.1.05/1.1.00/02.0123 from the European Regional Development Fund. 

References 

Guzi, T.J., et al.: Targeting the replication checkpoint using SCH 900776, a potent and 

functionally selective CHK1 inhibitor identified via high content screening. - Mol Cancer 

Ther,10(4): 591-602, 2011 

P33. THE EFFECTS TO MOLECULAR BIOMEMBRANE TRANSPORT HYPERFORIN OR 

ARISTOFORIN IN COLON ADENOCARCINOMA CELLS 

Martina Šemeláková, Rastislav Jendželovský, Jaromír Mikeš, Peter Fedoročko 

Institute of Biology and Ecology, Faculty of Sciences, P.J. Šafárik University in Košice, 

Slovak Republic; martina.semelakova@upjs.sk 

Photodynamic therapy is an anti-cancer approach for the treatment of various types 

of non-malignant as well as malignant diseases. Hypericin (HY), a naturally-occuring 

photosenzitive compound synthesized by Hypericum sp. (St. John’s wort), posses 

properties suitable for PDT and demonstrates photocytotoxic activity both in vitro and 

in vivo (Agostinis et al., 2000). Hyperforin is a phloroglucin-derivative that has emerged 

as a key player not only in the antidepressant activity of the plant’s extract but also as 

a suppressor of bacteria, lymphocyte and tumor cell proliferation (Erdelmeier, 1998), 

and as an inhibitor of matrix proteinases. Aristoforin, however, one of hyperforin’s 

synthetically prepared derivatives, has proved to be more soluble and even stable in 

aqueous solution. Importantly, it retains the antitumor properties of the parental 

compound without inducing toxicity in experimental animals. These data strongly 

suggest that AR has even greater potential as an anticancer drug (Gartner et al., 2005). 

Our previous results (Semelakova et al., 2012) showed that hypericin (HY)-mediated 

photodynamic therapy (HY-PDT) in sub-optimal dose with hyperforin (HP) (both bioactive 

compounds naturally occuring in plants of Hypericum sp.), or its stable derivative 

aristoforin (AR) were able to stimulate mechanisms leading to significant antitumor 

activity. Both, HP or AR were used at relatively low concentrations up to 5 μM. Significant 

accumulation of HY in cells after 16 h of incubation with drugs was described. 

Generally, the therapeutic efficacy of various chemotherapeutics is severely limited by 

drug resistance that is related to overexpression of drug efflux transporters in tumour 

cells. Therefore the impact of HY, HP or AR on cell’s ATP-binding cassette membrane 

transporter system, namely multidrug resistance-associated protein 1 (MRP1/ABCC1) 

and 2 (MRP2/ABCC2), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp/ 

MDR1) and cytochrome P450 monooxygenase, was subsequently evaluated by Western 

blot analysis of proteins, mRNA expression by qRT-PCR and flowcytometric analysis of 


proteins activity in HT-29 colon cancer cells. 

We report here decreased level of protein BCRP and decrease activity after treatment 

with 50 nM HY and HP or AR under the dark conditions after 16 h treatment, while no 

significant changes of MRP1, MRP2 and P-gp were found. When evaluating HY alone or 

combination of HY with AR (6 h after light-activation) decreased level of protein MRP2 

was observed. The therapy with light-activated HY modulated by 5 μM HP showed 

increased level of protein MRP1 and MRP2, in contrast to the decreased level of BCRP 

and MRP2 protein after therapy HY or AR alone or HY/5μM AR. However the most 

important analysis was transporter proteins activity which show decreased. 

The cytochrome P450 3A4 (CYP3A4) is one of the most important enzymes involved in the 

metabolism of xenobiotics in the human body. The protein level of CYP3A4 was increased 

by HY, HP and AR therapy alone or in combination of HY/1μM HP. The subsequent analysis 

of CYP3A4 proteins and mRNA 6 h after light-activation of HY therapy modulated by AR 

showed significant decreased level of protein, similarly decreased of mRNA expression 

and decreased of protein activity. We may conclude that pre-treatment with HP or AR 

affected proteins level of BCRP, MRP1 and MRP2, what could be one of the mechanisms 

responsible for pharmacokinetics of hypericin in HT-29 colon adenocarcinoma cells 

leading to higher therapeutic efficiency of HY-PDT. The inhibitory effect of HP and AR to 

activity of membrane transport proteins and to CYP3A4 protein also could be important 

as therapeutic strategies in the treatment of cancer. The presented data may help to 

elucidate the mechanisms of action for various bioactive constituents of St. John’s wort 

possessing potential therapeutic properties. 





References 

Agostinis, P., Assefa, Z., Vantieghem, A., Vandenheede, J.R., Merlevede, W., De Witte, P.: 

Apoptotic and anti-apoptotic signaling pathways induced by photodynamic therapy with 

hypericin. - Adv. Enzyme Regul. 40: 157–182, 2000. 

Erdelmeier, C.A.: Hyperforin possibly the major non-nitrogenous secondary metabolite 

of Hypericum perforatum L. – Pharmacopsychiatry 31: 2–6, 1998. 

Gartner M., T. Muller, J.C. Simon, A. Giannis, J.P. Sleeman, Aristoforin, a novel stable 

derivative of hyperforin, is a potent anticancer agent. - Chembiochem 6: 171–177, 2005. 

Semelakova, M., Mikes, J., Jendzelovsky, R. and Fedorocko, P.: The pro-apoptotic and 

anti- invasive effects of hypericin-mediated photodynamic therapy are enhanced by 

hyperforin or aristoforin in HT-29 colon adenocarcinoma cells. - Journal of Photochemistry 

and Photobiology B-Biology. 117: 115-125, 2012. 


P34. PHARMACOLOGICAL INHIBITION OF SKP2-SCF COMPLEX ACTIVITY AS AN 

APPROACH TO MODULATE CHARACTERISTICS OF CANCER STEM CELLS 

Šárka Šimečková 1,2* , Zuzana Pernicová 1,3 , Radek Fedr 1 , Alois Kozubík 1,2 , Karel Souček 1,3* 

1 


Republic, Brno, Czech Republic; 

2 



3 



*correspondence to: simeckova@ibp.cz, ksoucek@ibp.cz 

The cancer stem cells (CSCs) represent an attractive target for anticancer therapy. These 

cells are responsible for tumor relapse and serve as a reservoir of cancer cells within 

tumors. There are some characteristics of CSCs, which they share with their normal 

counterparts, as for example asymmetric division, differentiation into heterogeneous cell 

populations, and slow cycling. These cells have also an ability to invade into a surrounding 

tissue and importantly, they are more resistant to conventional drug treatment. 

Skp2 protein is a crucial component of Skp2-SCF complex that functions as an E3 

ubiquitin ligase involved in protein degradation. Skp2 is essential for cell cycle regulation 

via degradation of p27 Kip1 , Cdt1 and other molecules. High level of Skp2 was described 

in various types of cancer, including prostate and colorectal cancer. Pharmacological 

inhibition of Skp2-SCF complex has strong potential in anticancer therapy. Binding NEDD8 

to Cullins is necessary for Skp2-SCF formation and activity (Kawakami et al., 2001). 

Recently, a novel inhibitor of NEDD8 pathway MLN-4924 was described (Soucy et al., 

2009). Disruption in NEDD8 pathway using this inhibitor leads to accumulation of Skp2 

substrates and triggers cellular processes such as cell cycle arrest, cellular senescence 

and apoptosis. 

In this work we used MLN-4924 inhibitor and investigated its effect on murine 

adenocarcinoma cell line cE2 and human colorectal carcinoma cell line HCT116. 

Accumulation of Skp2-SCF substrates after MLN-4924 treatment led to deregulation of 

cell cycle in both cell lines. Interestingly, both cell lines express surface markers CD133 

and CD44 typical for CSCs and can be divided into two subpopulations based on the 

expression of CD133 – CD44 + CD133 low and CD44 + CD133 high (Fedr et al., 2013). Therefore, 

we further investigated the effect of MLN-4924 inhibitor on these subpopulations. MLN- 

4924 reduced clonogenic capacity of single cells in both cell lines and downregulated 

surface expression of CD133. Next, using flow cytometry we introduced a multicolor 

multiparametric protocol for simultaneous assessment of expression of surface CSCs 

markers, DNA synthesis, viability, cell cycle, apoptosis, and DNA damage. Using HCT116 

cells, we show that CD44 + CD133 low and CD44 + CD133 high subpopulations significantly 

differ in their response to treatment with MLN-4924 inhibitor. Higher sensitivity to DNA 

damage induction and arrest in G1 phase of the cells cycle was found in CD44 + CD133 low 

subpopulation, whereas CD44 + CD133 high subpopulation arrested preferentially in S phase 

and DNA damage was not observed. 


In summary, disruption of protein degradation pathway using MLN-4924 inhibitor is 

able to deregulate cell cycle in cE2 and HCT16 cell lines. Moreover, it affects stem cell 

properties like expression of CD133 and clonogenic capacity of the single cells. We also 

found different sensitivity of subpopulations CD44 + CD133 low and CD44 + CD133 high to MLN- 

4924 treatment. Thus, interestingly, this data show possible selective response of the 

cancer cells to the inhibition of neddylation based on their phenotype. 



and by project FNUSA-ICRC (no. CZ.1.05/1.1.00/02.0123) from the European Regional 



Reference 

FEDR, R., PERNICOVA, Z., SLABAKOVA, E., STRAKOVA, N., BOUCHAL, J., GREPL, M., 

KOZUBIK, A. & SOUCEK, K. (2013) Automatic cell cloning assay for determining the 

clonogenic capacity of cancer and cancer stem-like cells. Cytometry A. 

KAWAKAMI, T., CHIBA, T., SUZUKI, T., IWAI, K., YAMANAKA, K., MINATO, N., SUZUKI, H., 

SHIMBARA, N., HIDAKA, Y., OSAKA, F., OMATA, M. & TANAKA, K. (2001) NEDD8 recruits 

E2-ubiquitin to SCF E3 ligase. EMBO J, 20, 4003-12. 

SOUCY, T. A., SMITH, P. G., MILHOLLEN, M. A., BERGER, A. J., GAVIN, J. M., ADHIKARI, S., 

BROWNELL, J. E., BURKE, K. E., CARDIN, D. P., CRITCHLEY, S., CULLIS, C. A., DOUCETTE, 

A., GARNSEY, J. J., GAULIN, J. L., GERSHMAN, R. E., LUBLINSKY, A. R., MCDONALD, A., 

MIZUTANI, H., NARAYANAN, U., OLHAVA, E. J., PELUSO, S., REZAEI, M., SINTCHAK, M. 

D., TALREJA, T., THOMAS, M. P., TRAORE, T., VYSKOCIL, S., WEATHERHEAD, G. S., YU, J., 

ZHANG, J., DICK, L. R., CLAIBORNE, C. F., ROLFE, M., BOLEN, J. B. & LANGSTON, S. P. 

(2009a) An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. 

Nature, 458, 732-6. 

P35. SMALL MOLECULE SC3 – OLD STUFF, NEW WNT INHIBITOR 

Lucie Tůmová 1 , Antonio R. Pombinho 1 , Martina Vojtěchová 1 , Jitka Stančíková 1 , Dietmar 

Gradl 2 , Michaela Krausová 1 , Zbyněk Zdráhal 3 , Petr Bartůněk 1 and Vladimír Kořínek 1 

1 

Institute of Molecular Genetics, Academy of Science of the Czech Republic, Vídeňská 

1083, 142 20 Prague 4, Czech Republic; tumovalmg.cas.cz 

2 

Zoologisches Institut II, Universität Karlsruhe, Kaiserstrasse 12, 76131 Karlsruhe, 

Germany 

3 

Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, 

Czech Repubic 

The canonical Wnt signaling pathway is one of the crucial signaling cascades in cells, 

controlling cell proliferation and differentiation. Its non-physiological activation 

underlies variety of human diseases including many types of cancer, e.g. most discussed 

colorectal cancer. Therefore, inhibition of Wnt signaling became a prevalent theme 

in human cancer biology. Many studies have been previously reported, searching for 


novel chemotherapeutics with specific Wnt inhibitory effect. However, with such new 

compounds the examination of the metabolic stability and pharmacokinetics are 

necessary to be done before the engaging into preclinical studies. 

Compound SC3 is an antibiotic substance known since 80’s of the last century but only 

a little was reported about its molecular function within the cell. We identified this 

compound in our high-throughput screen for modulators of the canonical Wnt signaling 

pathway using Wnt/β-catenin responsive STF cells. The specificity and efficiency of SC3 

was subsequently confirmed in several in vitro and in vivo assays including TOPFLASH 

activity in luciferase assay and qRT-PCR in human colorectal cancer cell lines, double axis 

formation assay in Xenopus embryos and intestinal tumor treatment in APC Min mice. Cells 

cultivated with SC3 compound displayed reduced amount of β-catenin and in transient 

transfections, TOPFLASH activity of several constitutively active mutants of β-catenin 

was inhibited by the compound. These results suggest β-catenin-dependent effect of 

SC3 molecule, although the exact mechanism of action has not been elucidated yet. 

P36. PPARGAMMA IN COLON CANCER CELL DIFFERENTIATION AND DEATH INDUCED 

BY FATTY ACID TREATMENT 

Zuzana Tylichová 1,2 , Jiřina Hofmanová 1,2 , Pavel Krčmář 3 , Nicol Straková 1 , Alena Hyršlová 

Vaculová 1 , Alois Kozubík 1,2 

1 


Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic; 

2 

Department of Experimental Biology, Faculty of Science, Masaryk University, 611 37 


3 

Veterinary Research Institute,v.v.i. Brno, Czech Republic 

tylichova@ibp.cz 

Peroxisome proliferator-activated receptor γ (PPARγ) is nuclear receptor and transcription 

factor that is involved in regulating glucose and lipid homeostasis, inflammation, 

proliferation and differentiation. It is expressed mainly in colon and adipose tissues. 

Dietary exogenous as well as endogenous fatty acids and their metabolites function as 

PPARγ ligands and thus may affect many cellular events including cell cycle, fatty acid 

transport and utilization and important signaling pathways. PPARγ is often mutated in 

cancer cells which may contribute to cancer promotion and progression. 

Molecular mechanisms of PPARγ action were investigated in colon adenocarcinoma 

cell line HT-29 after treatment with short-chain fatty acid butyrate (NaBt) produced 

by microbial fermentation of fibre in the colon and essential ω-3 polyunsaturated 

docosahexaenoic fatty acid (DHA). Application of these coumpouds induced changes in 

PPARγ expression on gene and protein levels and also its significant activation (luciferase 

assay). The activation of PPARγ was additionaly supported using PI3K/Akt (Akt1/2) 

inhibitor which increased differentiation as well as cell death. Caspases inhibitor (zVADfmk) 

decreased paralelly PPARγ activation and cell differentiation but not apoptosis 

which indicates non-apoptotic function of caspases. Using flow cytometry and methods 


of molecular biology we detected changes of proliferation (CyQUANT), differentiation 

(alkaline phosphatase activity) and cell death (phosphatidylserine externalisation, 

caspase activation, mitochondrial membrane potential, PARP cleavage). These changes 

were accompanied by altered expression of some apoptotic (caspase 9) and autophagic 

(LC3, p62) proteins. Application of fatty acids also affected lipid metabolism regulating 

proteins (fatty acid synthase, caveolin-1) as well as cell ability to incorporate and store 

fatty acids (FAT/CD36, lipid droplet accumulation). 

In summary, our data suggest participation of PPARγ and specific signaling molecules 

in the effects of NaBt, DHA and their combination on colon cancer epithelial cell 

differentiation and death. 


This work was supported by grants Nos. P301/11/1730, 13-097665 of the Czech Science 

Foundation, NT 11205-5/2010 and MSM0021622430 Ministry of Education, Youth and 

Sports. 

P37. INHIBITORS OF CELLULAR ENERGY METABOLISM CAN ACTIVATE PRO-SURVIVAL 

SIGNALING IN MALIGNANT MELANOMA CELLS 

Amandine Verlande, Stjepan Uldrijan 

Department of Biology, Faculty of Medicine, Masaryk University Brno, Czech Republic 

Metastatic melanoma is a type of cancer that is notoriously difficult to treat, with only 

a minority of patients responding to standard chemotherapeutics. In a search for new 

drugs with anti-melanoma activity we investigated the effect of inhibitors of cellular 

energy metabolism on malignant melanoma cell survival and the activity of several 

important intracellular signaling pathways. 

We used three different inhibitors of mitochondrial oxidative phosphorylation (OXPHOS): 

Rotenone, CCCP and Oligomycin A, in combinations with two hexokinase inhibitors 

(glycolysis pathway): 2-deoxy-D-glucose (2DG) and 5-thio-D-glucose (5TG) in both BRAFand 

NRAS-mutated melanoma cells. 

Our results show that treatment with the OXPHOS inhibitors leads to cell death that 

can be prevented when 2DG or 5TG is added at low, non-toxic concentrations. We show 

that these drug combinations induce activation of AKT and AMPK pro-survival pathways. 

Even though AKT is strongly activated by the combinations, the mTORC1 complex seems 

to be inactive as we observed a simultaneous upregulation of autophagy and inhibition 

of protein synthesis. 

Taken together, these unexpected results suggest that the combination of OXPHOS and 

hexokinase inhibitors can activate pro-survival pathways in malignant melanoma and 

prevent cancer cell death. 


P38. EVIDENCE OF ABNORMAL PERIPHERAL CYTOKINE PROFILE IN PORCINE MODEL 

OF HUNTINGTON’S DISEASE 

Ivona Valekova 1,3 , Jiri Klima 1 , Helena Kupcova Skalnikova 2 , Karla Jarkovska 2 , 

Stefan Juhas 1 , Jan Motlik 1 

1 

Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and 

Genetics, v.v.i., AS CR, Libechov, Czech Republic; 

2 

Laboratory of Biochemistry and Molecular Biology of Germ Cells, Institute of Animal 

Physiology and Genetics, v.v.i., AS CR, Libechov, Czech Republic; 

3 

Department of Cell Biology, Faculty of Science, Charles University in Prague, Prague, 


valekova@iag.cas.cz 

Huntington´s disease (HD) is a devastating monogenic neurodegenerative disorder with 

detrimental effects of abnormal expansion of the CAG trinucleotide repeat within the 

coding region of the huntingtin gene. HD is characterized by brain neurodegeneration 

with a loss of brain neurons located predominantly in the striatum. The disease onset 

is in the middle age and is generally marked by progressive cognitive, psychiatric and 

motor impairment. Currently, there is no HD treatment available. 

Although HD causes widespread changes in CNS, there is a parallel immune activation in 

the periphery. The pro-inflammatory cytokine levels are increased earliest in the disease 

course, long before the onset of motor dysfunction. This phenomenon can be observed 

many years before the predicted onset of neurological symptoms (Björkvist et al., 2008), 

which designates immune activation as a potential biomarker of HD progression. 

The early disease progression is monitored on unique minipig model. In our lab we have 

recently generated a biomedical model of HD - a miniature pig transgenic for N-terminal 

part of the mutated human huntingtin (mt HTT, 548aa, 124Q) (Baxa et al., 2013). Agematched 

control and HD transgenic minipigs with the same genetic background were 

included in this study. 

The principal aims of this study are (i) to characterize the activation of innate immune 

system in control and HD transgenic animals, (ii) to monitor the levels of cytokines 

during ageing and disease course, (iii) to detect monocyte activation as a possible source 

of peripheral cytokines. 

The expression of selected thirteen cytokines, chemokines and growth factors were 

examined by targeted proteomic profiling using cytometric approaches. Isolated 

non-mature CD14+ monocytes were activated and analyzed using porcine Luminex 

immunoassay. 

Our data indicate significant changes between control and HD transgenic miniature 

pigs mainly in the level of IL-8, both in blood serum and monocytes in the age of 13- 

15 months. The cytokine profiling together with monitoring of monocyte activation is 

regularly continuing in three month intervals. 

Early changes identified in HD transgenic pigs may help to clarify the pathological 

mechanisms underlying the disease development, and thus could offer an obtainment 

of accessible non-invasive bioindicators of disease progress. 



This work is supported by the CHDI Foundation (ID 1035, A5378), The Technical 

Agency of the Czech Republic (TA01011466), RVO 67985904, and European Regional 

Development Fund CZ.1.05/2.1.00/03.0124. 

References 

Baxa, M. et al.: A Transgenic Minipig Model of Huntington´s Disease. – In: Journal of 

Huntington´s Disease 2(1). Pp. 47-68. 2013. 

Björkvist, M. et al.: A Novel Pathogenic Pathway of Immune Activation Detectable 

before Clinical Onset in Huntington´s Disease. – In: Journal of Experimental Medicine 

205(8). Pp. 1869-1877. 2008. 

P39. POTENTIATION OF HYPERICIN-MEDIATED PHOTODYNAMIC THERAPY TOXICITY 

BY 5-LOX PATHWAY INHIBITOR MK-886 

Barbora Valeková, Jaromír Mikeš, Rastislav Jendželovský, Ján Kovaľ, Jana Vargová, Lucia 

Mikešová, Peter Fedoročko 


Košice, Slovak Republic; barbora.valekova@student.upjs.sk 

Arachidonic acid and its metabolites play significant role in cancer biology. Since interfering 

with arachidonic acid metabolism may result in cancer cell growth, invasiveness and 

angiogenesis arrest, inhibitors of cyclooxygenase, lipoxygenase and cytochrome P450 

monooxygenase pathways of this metabolic cascade, nonsteroidal anti-inflammatory 

drugs, are attracting attention as potent anticancer agents. There is growing evidence 

that these therapeutics exhibit also cytotoxic and antiproliferative effects via pathways 

independent of inhibiting arachidonate metabolization. Various target molecules were 

described, including GDF-15, a controversial cytokine. Its increased expression and 

accumulation in cancer cells in various models correlates with activation of apoptotic 

pathways decrease in cell proliferation and survival, but exact role of GDF-15 in these 

processes remains undefined (Pang et al., 2007). 

Photodynamic therapy is an anticancer treatment modality based on activation of 

photosensitizer localized within tumour cells by light of appropriate wavelength in the 

presence of oxygen. Consequent photophysical and photochemical reactions create 

reactive oxygen species that damage the target cells and induce cell death pathways. 

However, photodynamic therapy can lead to arachidonic acid release via activation of 

phospholipases C and A 2 

and its metabolization to eicosanoids that promote prosurvival 

and proliferation signals (Agarwal, 1993). Modulation of arachidonic acid metabolism is 

therefore a promising approach to increase efficiency of photodynamic therapy. 

As a photosensitizer for our experiments we used hypericin, a naturally occurring 

naphtodianthrone with high affinity to cancer cells, minimal dark toxicity and high 

efficiency in oxidative stress induction (Agostinis et al., 2002). To modulate arachidonic 

acid metabolism we chose MK-886. It suppresses 5-lipoxygenase pathway by interaction 

with active site of 5-lipoxygense activating protein, preventing presentation of arachidonic 


acid to 5-lipoxygenase. Similarly to other nonsteroidal anti-inflammatory drugs, MK-886 

is characterized by activities independent of lipoxygenase inhibition (Datta et al., 1999). 

In our experiment, 48 hour pre-treatment with various concentrations of MK-886 

prior to hypericin activation in HT-29 human colorectal adenocarcinoma cell line led to 

significant decrease in metabolic activity compared to individual treatments. Impairment 

of metabolic activity was accompanied by cell viability reduction, mitochondrial 

membrane potential disruption and massive caspase-3 activation, indicating apoptotic 

form of cell death. Cells pre-treated with MK-886 also showed significant increase in 

hypericin intracellular accumulation. These results together with observed changes in 

redox systems may represent 5-lipoxygenase independent actions of MK-886 in cancer 

cells. We also detected progressive accumulation of pleiotropic cytokine GDF-15 in HT- 

29 cells after treatment with MK-886 or hypericin-mediated photodynamic therapy 

alone and combined treatment. GDF-15 accumulation correlated with evaluated cell 

death markers. However, the role of GDF-15 in action of MK-886, hypericin-mediated 

photodynamic therapy and their mutual combination was not confirmed and its 

accumulation is considered to be a marker of cell damage. 





References 

Agarwal, M.L., Larkin, H.E., Zaidi S. I., Mukhtar, H. and Oleinick, N.L.: Phospholipase 

activation triggers apoptosis in photosensitized mouse lymphoma cells. - Cancer 

Research 53: 5897–5902, 1993. 

Agostinis, P., Vantieghem, A., Merlevede, W. and de Witte, P.A.M.: Hypericin in cancer 

treatment: more light on the way. - Cell Biology 34: 221-241, 2002. 

Datta, K., Biswal, S. S., Kehrer, J. P.: The 5-lipoxygenase-activating protein (FLAP) inhibitor, 

MK886, induces apoptosis independently of FLAP. – Biochemical Journal 340: 371–375, 

1999. 

Pang, R., Zhou, J., Zeng, Z., Li, X., Chen, W. and Hu, P.: Celecoxib induces apoptosis in 

COX-2 deficient human gastric cancer cells through Akt/GSK3b/NAG-1 pathway. - Cancer 

Letters 251: 268–277, 2007. 

P40. HISTAMIN AND ITS H 4 

RECEPTOR AGONISTS DECREASE THE REACTIVE OXYGEN 

SPECIES PRODUCTION IN HUMAN LEUKOCYTES AND ACT AS CHEMOATTRACTANTS 

O. Vasicek 1,2 , M. Ciz 1 , A. Lojek 1 

1 

Institute of Biophysics, Academy of Sciences of the Czech Republic, v. v. i., Kralovopolska 

135, 612 65 Brno, Czech Republic, ondrej.vasicek@ibp.cz 

2 

Department of Animal Physiology, Institute of Experimental Biology, Faculty of Science, 

Masaryk University, Kotlářská 267/2, 611 37 Brno, Czech Republic 

Histamine, an endogenous biogenic amine, is an important chemical messenger which 


has numerous physiological roles in central and peripheral tissues [1,2]. These effects are 

mediated through four histamine receptors (H 1 

R, H 2 

R, H 3 

R and H 4 

R) which belong to the 

superfamily of G protein-coupled receptors [3]. We investigated the effects of histamine 

and the H 4 

R agonists 4-methylhistamine and VUF8430 on the production of reactive 

oxygen species (ROS) and chemotaxis in human whole blood and isolated leukocytes. 

The antioxidant properties of histamine receptor agonists were investigated using total 

peroxyl radical-trapping antioxidant parameter analysis, oxygen radical absorbance 

capacity assay and NO-scavenging determination. ATP activity was used for evaluation 

of cell viability. The ability of isolated leukocytes or leukocytes in the whole blood of 

healthy human volunteers to produce ROS after histamine or H 4 

R agonist treatment (10 - 

8 

-10 -4 M) was tested by luminol-enhanced chemiluminescence, spontaneous or activated 

by opsonised zymosan particles (OZP) or phorbol-myristate-acetate (PMA). Further, 

Dimaprit (H 2 

R agonist), Ranitidine (H 2 

R antagonist) and JNJ10191584 (H 4 

R antagonist) 

were used for confirmation of histamine effects mediated through different types of 

histamine receptors. Confocal microscopy was used for chemotactic measurements. 

None of the studied compounds had any antioxidant activity against ROS. H 4 

R agonists 

significantly decreased the spontaneous and OZP-activated chemiluminescence 

response in whole blood leukocytes in a dose-dependent manner. In contrast, only 

VUF8430 was dose-dependently effective when whole blood leukocytes were activated 

with PMA. Generally, the effects of all three compounds were similar but less profound 

in isolated leukocytes. Ranitidine more than JNJ10191594 averted the inhibition of ROS 

production by histamine or his agonists. H 4 

R agonists had a positive chemotactic effect 

on the isolated leukocytes, but not directly on neutrophils. 

It can be concluded that the inhibition of ROS production by the tested compounds was 

caused by H 2 

R rather than by H 4 

R activation. The specificity of the H 4 

R agonists tested is 

dependent on the concentration used and, especially at high concentrations, the signal 

may be transduced mainly through H 2 

R. We assume from our results that neutrophils do 

not express active H 4 

R. 


Supported by grant LD11010 of the Ministry of Education, Youth and Sports of the Czech 

Republic and by COST BM0806 Action. 

1. Shahid, M., T. Tripathi, F. Sobia, S. Moin, M. Siddiqui, and R. Khan, Histamine, histamine 

receptors, and their role in immunomodulation: an updated systematic review. The Open 

Immunol. J., 2009. 2: p. 9-41. 

2. Zampeli, E. and E. Tiligada, The role of histamine H4 receptor in immune and 

inflammatory disorders. Br J Pharmacol, 2009. 157(1): p. 24-33. 

3. Oda, T., N. Morikawa, Y. Saito, Y. Masuho, and S. Matsumoto, Molecular cloning 

and characterization of a novel type of histamine receptor preferentially expressed in 

leukocytes. J Biol Chem, 2000. 275(47): p. 36781-6. 


P41. WHY CAN WE SEE DIFFERENT RADIOSENSITIVITY OF LYMPHOCYTE SUBSETS? 

Lenka Zárybnická 1 , Zuzana Šinkorová 1 , Zuzana Kročová 2 , Jiřina Vávrová 1 

1 

Department of Radiobiology, Faculty of Military Health Sciences, University of Defence, 

Hradec Králové, Czech Republic; zarybnicka@pmfhk.cz 

2 

Institute of Mollecular Pathology, Faculty of Military Health Sciences, University of 

Defence, Hradec Králové, Czech Republic 

Lymphocytes exposed to gamma-ray irradiation experience a degradation of their 

macromolecules, especially DNA. Cells unsuccessful in DNA repairing process 

induce apoptosis. Analysis of apoptotic peripheral blood mononuclear cells (PBMC) 

by phosphatidyl serine detection (Annexin-V antibody) after in vitro irradiation is 

dose dependent thus PMBC can be used as in vitro sensitive biodosimetric markers 

(Šinkorová et al. 2011). Nevertheless, it is not possible to detect the apoptotic fraction 

by Annexin-V antibody after in vivo irradiation (our unpublished results) due to the early 

in vivo elimination of apoptotic cells from peripheral blood by active scavenging system 

(Bogdandi et al. 2010), thus only the not influenced cells or repaired cells can be in vivo 

studied. Many of studies confirmed different radiosenzitivity of individual lymphocyte 

subsets where especially B lymphocytes are the most radiosensitive (Girinsky et al. 

1991). 

In this work we studied the radiosenzitivity of peripheral blood T-lymphocytes, 

B-lymphocytes and natural killers (NK cells). We were looking for changes between 

individual lymphocyte subsets after in vivo irradiation ( 60 Co, gamma source) focusing 

on the very early phase of apoptosis when a decrease of mitochondrial membrane 

potential ( D 

ψ) occurs and furthermore on the period which precede the apoptosis, the 

cell repair process. At the site where DNA is damaged the DNA double-strand breaks 

(DSB) are produced and DNA damage repair factors are accumulated on chromatin. 

One of the key processes activated within minutes after the DSB induction is the 

phosphorylation of histone H2AX at serine 139 (γ-H2AX). γ-H2AX expression assessment 

has been successfully exploited as in vivo biodosimetric marker (Rothkamm and Horn 

2009). Mitochondrial membrane potential changes occuring within very early phases of 

apoptosis can be analyzed by D 

ψ-sensitive probe (JC1) which has been successfully used 

within many apoptotic studies. We established new protocols combined intracellular 

detection of γ-H2AX, respective D 

ψ changes, with extracellular immunophenotyping 

surface markers specific for rat or porcine NK cells (CD8, CD161), T-lymphocytes (CD3, 

CD4, CD8) and B-lymphocytes (CD45RA) which were analyzed by flow cytometry 

(CyAn ADP, Beckman Coulter). Using the animal experimental models (Wistar rats and 

large white pigs) which were influenced by in vivo irradiation the changes of γ-H2AX 

expression, respective D 

ψ decrease, within lymphocyte subsets were analyzed. Studies 

were approved by the Ethical Committee of the Faculty of Military Health Sciences in 

Hradec Kralove and by the Ethical Committee of the Ministry of Defence of the Czech 

Republic. 

In our previous studies we confirmed that each lymphocyte subset within both animal 

models (porcine, rat) exerts its own characteristic in vivo radiosenzitivity. In this study, 


a very significantly increased γ-H2AX expression was detected in rat B-lymphocytes. 

Differences between T-lymphocytes and NK cells were evident after a high dose (9 Gy) 

and revealed the least intensive response in CD161 NK cells. The increase in all subsets 

was dose-dependent (Zárybnická et al. 2013). In contrary, we found out that it is not 

possible to detect D 

ψ decrease in porcine lymphocytes after in vivo irradiation instead 

of the fact that these changes are connected with very early phases of apoptosis. Hence 

we analyzed D 

ψ changes within in vitro irradiated lymphocytes where no significant 

differences between T-lymphocytes and NK cells were found (B-lymphocytes were 

not studied). Results show dose dependency upon 0 – 2 Gy. The apoptotic fraction for 

higher doses is stable nevertheless representation of this fraction increases with in vitro 

cultivation time. 

Peripheral blood lymphocytes are generally used as sensitive biodosimetric markers of 

in vivo irradiation. Different radiosenzitivity of individual lymphocyte subsets propounds 

the question what are differences between DNA double strand breaks repairing and/ 

or apoptosis inducing between of these subsets. Analysis of γ-H2AX expression and D 

ψ 

changes is only first step in this study. The advanced biodosimetric approach combining 

lymphocyte surface immunophenotyping with intracellular detection suggests the 

possibility for exploitation for the future development of a biodosimetry procedure. 


This work was supported by the Ministry of Defence of the Czech Republic (The 

institutional support to a long-term organization development plan 1011, project No. 

OVUOFVZ200806 and project No. OVUOFVZ200809). 

References 

Bogdándi EN, Balogh A, Felgyinszki N, Szatmári T, Persa E, Hildebrandt G, Safrany G, 

Lumniczky K. Effects of low-dose radiation on the immune system of mice after totalbody 

irradiation. Radiation Research 174: 480-489. 2010. 

Girinsky T, Socie G, Cosset JM, Malaise EP. Blood lymphocyte subsets after the first 

fraction in patients given hyperfractionated total body irradiation for bone marrow 

transplantation. British Journal of Cancer 63: 646-647. 1991. 

Rothkamm K, Horn S. Gamma-H2AX as protein biomarker for radiation exposure. Annali 

Dell Instituto Superiore di Sanita 45: 265-271. 2009. 

Šinkorova Z, Sinkora J, Zarybnicka L, Vilasova Z, Pejchal J. Radiosensitivity of peripheral 

blood B cells in pigs. Veterinarni Medicina 54: 223-235. 2009. 

Zárybnická L, Vávrová J, Havelek R, Tichý A, Pejchal J, Šinkorová Z. Lymphocyte subsets 

and their H2AX phosphorylation in response to in vivo irradiation in rats. Internatinal 

Journal of Radiation Biology. 89(2): 110-7. 2013. 


P42. ELUCIDATION OF CROSSTALK BETWEEN SENESCENCE AND APOPTOSIS 

Zuzana Nahacka, Gita Novakova, Ladislav Anděra 

Institute of Molecular Genetics of the Academy of Science of Czech Republic, Laboratory 

of Cell Signaling and Apoptosis, Prague, Czech Republic, nahackaz@img.cas.cz 

Senescence is irreversible cell-cycle arrest, where senescent cells stay metabolically 

active and acquire distinctive morphological and biochemical alterations (Bartek et al., 

2007). Replicative senescence was described in normal cells as an intrinsic molecular 

programme that limits cell multiplication (Hayflick and Moorhead, 1961). Apart from 

aging, senescence in normal human cells can be induced by oncogenes such as activated 

H-Ras as a protective measure against tumorigenesis. Furthermore stress-induced or 

premature senescence is one of the major cellular response to chemotherapy and 

radiotherapy in solid tumors (Mirzayans et al., 2005). 

The other type of cell protection against tumorigenesis is apoptosis. In our study we are 

mainly interested in an elucidation of presumed crosstalk between apoptotic signaling 

and senescence-triggered rewiring the cellular signaling and metabolism. 

We induced premature senescence in primary (colorectal NCM460) and cancer cell 

lines (pancreatic PANC and mestohelial H28) with BrdU (20-100uM) treatment for 5-7 

days. Then using mainly flow cytometry we analyzed response of senescent cells to 

different apoptotic stimuli inducing extrinsic (Trail, Fas ligand) or intrinsic (mito-VES, 

staurosporine, campthotecin) apoptotic signaling and the expression profile of TRAIL-R1 

(Death receptor 4), TRAIL-R2 (Death receptor 5), TRAIL-R3 (Decoy receptor 1), TRAIL-R4 

(Decoy receptor 2), FAS and TNF receptors. In addition to drug-induced senescence 

we are currently testing recombinant lentiviruses with inducible (pLVX p16-T2A-p21) 

or constitutive (pCDH p16-T2A-p21), expression of cell cycle inhibitors p16 and p21 

proteins. We found that Fas expression has increased in the drug-induced senescent 

cells which is in agreement with recently published data (Crescenzi et al., 2011). These 

cells were also sensitized to FasL-induced apoptosis. Similarly, these senescent cells 

were sensitized to campthotecin and mito-VES triggered apoptosis. In contrast, DR4 

expression and sensitivity to TRAIL dropped in the senescent cells. 

References 

Hayflick, L. and Moorhead, P.S. (1961) The serial cultivation of human diploid cell strains. 

Exp Cell Res, 25, 585-621. 

Mirzayans, R., Scott, A., Cameron, M. (2005) Induction of accelerated senescence by 

gamma radiation in human solid tumor-derived cell lines expressing wild-type TP53. 

Radiat Res, 163, 53-62. 

Bartek, J., Bartkova, J., Lukas, J. (2007) DNA damage signalling guards against activated 

oncogenes and tumour progression. Oncogene, 26, 7773-7779. 

Crescenzi, E., Pacifico, F., Lavorgna, A. et al. (2011) NF-κB-dependent cytokine secretion 

controls Fas expression on chemotherapy-induced premature senescent tumor cells. 

Oncogene, 30, 2707-2717. 


P43. HIERARCHICAL CLUSTER ANALYSIS OF 14-PARAMETER FLOW CYTOMETRY 

IMMUNOPHENOTYPING IDENTIFIES SHIFTS IN THE CIRCULATING T-CELL 

COMPARTMENT FOLLOWING REPERFUSION IN PATIENTS WITH ACUTE MYOCARDIAL 

INFARCTION. 

Karel Fišer 1 , Jedrzej Hoffmann 2 , Jolanta Weaver 4 , Ian Dimmick 2 , Monika Loeher 2 , 

Hanspeter Pircher 5 , Carmen Martin-Ruiz 3 , Murugapathy Veerasamy 2 , Bernard Keavney 2 , 

Thomas von Zglinicki 3 , Ioakim Spyridopoulos 2 

1 

CLIP – Childhood Leukaemia Investigation Prague, Department of Paediatric 

Haematology and Oncology, 2 nd Faculty of Medicine, Charles University and University 

Hospital Motol, Prague, Czech Republic, karel.fiser@lfmotol.cuni.cz 

2 

Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom, 

3 

Institute of Ageing and Health, Newcastle University, Newcastle, United Kingdom, 

4 

Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom, 

5 

Department of Immunology, Institute of Medical Microbiology and Hygiene, Freiburg 

University, Freiburg, Germany 

In patients with acute ST-elevation myocardial infarction (STEMI) reopening of the infarct 

related coronary vessel by primary percutaneous coronary intervention (PPCI) is the 

most effective treatment strategy. However it bears its risks as the following myocardial 

reperfusion can induce myocardial injury and cell death. Therefore, future therapies 

for STEMI have to target causes of post-infarction heart failure, including myocardial 

reperfusion injury. 

We used 14-parameter flow cytometry immunophenotyping to investigate the 

involvement of T cell subtypes in reperfusion after PPCI. Using hierarchical cluster 

analysis (HCA) we identified a unique CD4(+)CD57(+) T-cell population in PPCI patients 

that reflected acute proliferation in the CD4(+) T-cell compartment. We also marked 

contraction of effector T cells in STEMI patients with CMV positivity, possibly contributing 

to development of immunosenescence in these patients. 

In summary, high-throughput polychromatic flow cytometry and HCA are capable of 

objective, time and cost efficient assessment of the individual T-cell immune profile in 

different stages of coronary heart disease and have broad applications in clinical trials. 


This work was supported by University research centre (UNCE) grant, UNCE 204012. 


P44. INSTRUMENT SETTINGS FOR EUROFLOW STANDARDIZED 8-COLOR PANELS ON 

DIFFERENT FLOW CYTOMETRY PLATFORMS 

Michaela Nováková 1 , Marcela Vlková 2 , Daniel Thürner 1 , Juan Flores Montero 3 , Mikael 

Roussel 4 , Ana Helena Santos 5 , Ester Mejstříková 1 , Quentin Lecrevisse 3 , Ondřej Hrušák 1 , 

Tomáš Kalina 1 

1 


Prague 5, Czech Republic, michaela_novakova@lfmotol.cuni.cz 

2 

Department of Clinical Immunology and Allergology, St. Anne’s University Hospital and 


3 

Cancer Research Center (IBMCC-CSIC), Department of Medicine and Cytometry Service, 

University of Salamanca, Salamanca, Spain 

4 

Hematology Laboratory, CHU Pontchaillou, Rennes, France 

5 

Centro Hospitalar do Porto, Portugal 

Background: EuroFlow consortium has recently developed a standardized approach to 

immunophenotyping of hematological malignancies (Kalina et al., 2012; van Dongen et 

al., 2012). The standardization is performed on both levels, uniform antibody panels 

and uniform instrument settings, so that a computational meta-analysis of the data is 

possible. However, due to availability of the instruments at the project’s beginning in 

2006, we have proven the approach only on 8-color BD Biosciences digital instruments. 

Lately, 8-color flow cytometry has become available on several flow cytometry platforms 

of different makers. Thus, we tested the feasibility of standardized acquisition and 

merged analysis of data across the platforms. 

Methods: We have set the PMT voltage using hard-dyed 8-peak Rainbow beads to reach 

common target channel values. Where needed, target values were re-scaled to 18-bit 

common scale. Since the fluorochromes used in Rainbow beads do not completely 

correspond to fluorochromes used in Lymphocytosis Screening Tube, we used 2 types 

of capture beads stained with actual monoclonal antibodies to achieve more accurate 

target values for different instruments. 

We have stained peripheral blood of three healthy donors with modified EuroFlow 

Lymphocytosis Screening Tube to obtain discrete positive lymphocyte subset in all 

8 channels. After staining we split the samples and acquired on BD FACS Canto II, BC 

Navios, BC Cyan ADP and Miltenyi MACSQuant Analyzer. Next, we re-scaled to 18-bit, 

merged and analyzed in Infinicyt software. 

Improved target values were confirmed by measuring three peripheral blood samples 

from healthy donors in 5 Euroflow centers (Prague, Brno, Salamanca, Rennes, Porto) on 

3 types of instruments (5 BC Navios, 3 BD Canto II, 3 Dako Cyan). 

Results: Data obtained from the same sample on different cytometry platforms were 

very similar. After gating lymphocytes on FSC and SSC (scatter parameters were not 

standardized), we could apply the same gating strategy (gate position) on all samples. 

When we analyzed MFI of gated positive subsets, the values were distributed with 

average CV of 18,8% (range 7% - 32%), which presents variability that is lower than both 

the inter-individual and inter-laboratory variability based on the previous quality control 


ounds on BD instruments only. Background staining levels (negative lymphocytes) were 

also comparable. While we did see systematic platform related differences when using 

hard dyed Rainbow beads, probably attributed to different filter sets used in collection 

optics, they were improved by using single stained capture beads. 

Conclusions: We show that polychromatic flow cytometry protocols can be standardized 

even across different flow cytometry platforms developed by different makers. This opens 

an opportunity to data exchange, inter-laboratory collaborations and computational data 

analysis of multi centric studies regardless of flow cytometry equipment used. Variability 

of the data introduced by instrument is lower than sample preparation variability. 


Supported by UNCE 204012, GAČR P/302/12/G101, GAČR P/301/10/1877, NT/12425-4, 

NT/14534 

References 

Kalina T. et al: EuroFlow standardization of flow cytometer instrument settings and 

immunophenotyping protocols. Leukemia. 2012 Sep;26(9):1986-2010. 

van Dongen J.M.M. et al: EuroFlow antibody panels for standardized n-dimensional flow 

cytometric immunophenotyping of normal, reactive and malignant leukocytes. 

Leukemia. 2012 Sep;26(9):1908-75. 

P45. USE OF MICROFLUIDICS TO ACHIEVE NATIVE PHENOTYPE OF ENDOTHELIAL CELLS 

Barbora Ambrůzová 1,2 , Hana Kolářová 1,3 , Martin Kubeš 1,2 , Lucia Binó 1,2 , Jan Víteček 1,3 , 

Lukáš Kubala 1,3 

1 

Institute of Biophysics ASCR v.v.i., Brno, Czech Republic; 258462@mail.muni.cz 

2 


Republic; 

3 

International Clinical Research Center – Centre of Biomolecular and Cellular 

Engineering, St. Anne‘s University Hospital Brno, Czech Republic 

Vasculature is a complex system composed of many cell types organized in a specific 

manner to fulfill its physiological function. Vascular endothelial cells play role in many 

physiological processes, e.g. maintenance of vascular tone and immune response (Choi, 

2009; Giles, 2012). 

The native phenotype of these cells is characterized by elongated shape, organization in 

the direction of blood flow, tight contacts among them and remarkable glycocalyx layer 

at the luminal surface. Such phenotype is however suppressed under standard static in 

vitro cultivation (Henderson-Toth, 2012). 

Our main focus is the inner layer of endothelial cells. In order to get native phenotype 

of endothelial cells, HUVECs were cultivated under shear stress in a flow through system 

operated by IBIDI pump. Flow conditions resulted in elongation of cells and longitudinal 

orientation with flow. Further, elevated expression of glycocalyx-related genes was 

detected using RT PCR after 3 and 7 days. 

It can be suggested that microfluidic system based on IBIDI pump and µ-slides can 


e used to cultivate endothelial cells in order to induce phenotype similar to in vivo 

conditions in blood vessels. 


This work was supported by the European Social Fund - Project OrganoNET 

(CZ.1.07/2.4.00/31.0245) and HistoPARK (CZ.1.07/2.3.00/20.0185) and project FNUSA- 

ICRC (CZ.1.05/1.1.00/02.0123). 

References 

Choi, E.Y., Santoso, S., Chavakis, T.: Mechanisms of neutrophil transendothelial migration. 

– Front Biosci 1: 1596-1606, 2009. 

Giles, T.D., Sander, G.E., Nossaman, B.D., Kadowitz, P.J.: Impaired vasodilatation in the 

pathogenesis of hypertension: focus on nitric oxide, endothelial-derived hyperpolarizing 

factors, and prostaglandins. – J Clin Hypertens (Greenwich) 14: 198-205, 2012. 

Henderson-Toth, C.E., Jahnsen, E.D., Jamarani, R., Al-Roubaie, S., Jones, E.A.: The 

glycocalyx is present as soon as blood flow is initiated and is required for normal vascular 

development. – Dev Biol 15: 330-339, 2012. 

P46. DEVELOPMENT OF EUROFLOW STANDARDIZED PROTOCOL FOR DIAGNOSTIC 

EVALUATION OF PRIMARY IMMUNODEFICIENCY 

Tomáš Kalina 1 , Vendula Šinkorová 1 , Ester Mejstříková 1 , Michaela Nováková 1 , Marcela 

Vlková 2 , Menno C. van Zelm 3 , Martin Perez-Andres 4 , Eduardo Lopez 5 , Alberto Orfao 4 , 

Jacques J. M. van Dongen 3 , Mirjam van der Burg 3 

1 


Praha 5, Czech Republic, 

2 

Department of Clinical Immunology and Allergology, St. Anne’s University Hospital and 


3 

Immunology Department, Erasmus MC, Rotterdam, The Netherlands 

4 

Cancer Research Center (IBMCC-CSIC), Department of Medicine and Cytometry Service, 

University of Salamanca, Salamanca, Spain 

5 

Clinical Immunology Service, Hospital La Paz, Madrid, Spain 

Primary immunodeficiency (PID) is an immune system malfunction caused by hereditary 

mutation of genes encoding proteins which are involved in differentiation or effector 

functions of immune cells or immunodeficiency with unknown cause (not related to 

infection or therapy) . Flow cytometric immunophenotyping of peripheral blood and 

bone marrow has become central in diagnostics and research on PID. Many centers have 

developed multi-color flowcytometric protocols, but differences in antibody panels, 

sample handling, instrument setup and data analysis hamper exchange of data between 

centers and understanding of rare cases. 

The goal of EuroFlow PID project was to develop standardized 8-color immunophenotyping 

panel for initial screening of patients suspected with immunodeficiency. First tube (so 

called “screening tube”) of this panel allows for analysis of all main lymphocyte subsets 

(T, B and NK cells) in one tube and their maturational status. Standardized, multi centric 

approach facilitates joint data analysis using novel data analysis tools (Infinicyt software). 


We have tested optimal composition and performance of the screening tubes over 4 

modifications of 8-color panel on the total of 270 peripheral blood samples from patients 

and healthy donors. Of them, there were 29 patients with molecularly defined mutation 

and 56 patients with Common variable immunodeficiency (CVID). We have reached 

concordance with standard 4-color tubes. Major perturbations (absence of lymphocyte 

subset) were correctly detected in Severe combined immunodeficiency (SCID) and 

Cartilage-hair hypoplasia (CHH). Maturation perturbations, such as absence of memory 

B cells or memory T cells were detected in Hyper IgM syndrome (HIGM), Wiskott-Aldrich 

syndrome (WAS), DiGeorge syndrome (DGS) and CVID. Dedicated 8-color tubes focused 

on maturational stages of T or B cells further investigated individuals with abnormal 

results of the EuroFlow PID screening tube. 

EuroFlow PID screening tube allowed us to detect abnormalities in all major lymphocyte 

subsets. Standardization of the sample preparation protocols and data acquisition allows 

for multi-centric evaluation of the rare diseases and will also enable sharing anonymized 

phenotype data to facilitate diagnostic process and expand our understanding of cellular 

immunity in PID. 

Acknowledgements: Supported by grant NT/13271, NT/13287-4, NT/11414-5 

P47. THE USE OF FLOW CYTOMETRY IN THE SELECTION OF PROBIOTIC BACTERIA 

Dagmar Mudronova, Radomira Nemcova, Dana Ryznerova 

Department of Microbiology and Immunology, University of Veterinary Medicine and 

Pharmacy, Kosice, Slovak Republic; mudronova@uvlf.sk 

Flow cytometry is mostly used for identification, counting and testing of some properties 

of eukaryotic cells. In recent years, however, increasingly being used in microbiology, 

especially for counting and viability testing of bacteria. Any potentially successful probiotic 

bacteria designated for oral administration must fulfill some selection criteria. They 

must be able to survive and grow in the gastrointestinal tract conditions and to adhere 

to the mucosa of the gut. It is also necessary to respect the origin of the strain used and 

its ability to inhibit pathogens. The strain should be precisely identified, safe, genetically 

stable, it should have good growth properties, to maintain its high viability at processing 

and when in storage. Depending on the desired outcome, a probiotic strain may need to 

have additional properties, such as anticarcinogenic or hypocholestrerolemic effects, or 

the ability to improve lactose utilization (Nemcová, 1997). In the first phase of selection, 

most of the properties are tested in in vitro conditions. For tests on the gastrointestinal 

tract and technological process survival are often used conventional, time- and materialconsuming 

microbiological methods. An alternative, rapid and reliable technique for 

viability assessment seems to be flow cytometry (Bunthof et al., 1999). The aim of this 

study was to investigate the survival of beneficial bacteria in simulated gastrointestinal 

conditions and during the technological process by flow cytometry. 

For the simulation of the gastrointestinal tract conditions was prepared artificial gastric 

juice with pH adjusted to different levels, intestinal juice and bile salts solutions with 


concentrations 0.15, 0.2, 0.3 and 0.5 %. From the technological procedures used during 

the cheese production have been tested the effects of temperature (58 and 70 ° C) and 

high concentration of NaCl (5 M). Two strains of lactobacilli were used for the trials - 

exopolysaccharides (EPS) producing strain L. reuteri and EPS non-producing L. casei. To 

assess the viability of lactobacilli in the gastric juice, in a NaCl solution and under the 

influence of temperature was used propidium iodide (PI) staining. Due to the non-specific 

reaction between PI and bile salts viability was assessed by the help of carboxyfluorescein 

diacetate staining in the intestinal juice and bile salts. The percentages of viable bacteria 

were evaluated on the basis of SSC vs. Fl-3 histograms for PI, and SSC vs. Fl-1 histograms 

for cFDA. 

Survival of EPS-producing L. reuteri at pH 2 and 2.5 of gastric fluid, in the intestinal 

juice, in the presence of bile salts at concentrations 0.15, 0.2 and 0.3 %, and 5 M NaCl 

solution was significantly higher in comparison with EPS non-producing L casei. EPS 

production did not affect survival of strains at high temperatures, in the presence of very 

high concentration of bile salts (0.5 %), even at extremely low pH of gastric juice (pH 

1.5). The experiment results confirmed the protective effect of exopolysaccharides on 

bacteria to adverse external conditions. Flow cytometry is suitable and reliable method 

for assessment of bacterial viability in different conditions important for the selection 

of probiotic bacteria, but appropriate fluorescent dyes must be used for different tests 

since reactions between fluorochromes and used chemicals can occur. 


This study was supported by the project of the Slovak Research and Development Agency 

- APVV-0199-11 and the project VEGA 1/0834/12. 

References 

Nemcova R: Selection criteria of lactobacilli for probiotic use. - Vet Med Czech 42:19-27, 

1997. 

Bunthof C. J., van den Braak S., Breeuwer P., Rombouts F. M., Abee T. Rapid fluorescence 

assessment of the viability of stressed Lactococcus lactis. - Appl Environ Microbiol 65: 

3681-3689, 1999. 

P48. NILE RED STAINING AND FLOW CYTOMETRY ANALYSIS OF 

POLYHYDROXYBUTYRATE (PHB) ACCUMULATING BACTERIAL CELLS 

Stanislav Obruca 1 , Dan Kucera 2 , Ivana Marova 1,2 

1 

Centre of Materials Research, Faculty of Chemistry, Brno University of Technology, 

Brno, Czech Republic; e-mail: obruca@fch.vutbr.cz 

2 

Department of Food Chemistry and Biotechnology, Faculty of Chemistry, Brno 

University of Technology, Brno, Czech Republic 

Polyhydroxybutyrate (PHB) is polyesters accumulated by various bacteria in form of 

intracellular granules which serve as a carbon and energy reserve material. Generally, 

PHB is synthetized when carbon source is present in excess and bacteria cells are limited 

by availability of nitrogen and/or phosphorous sources. Due to its mechanical and 


technological properties PHB is considered as an alternative to petrochemical polymers 

such as polyethylene or polypropylene. Moreover, unlike synthetic polymers, PHB can 

be produced from renewable or waste substrates and it is also fully biodegradable and 

biocompatible (Kessler and Wilholt 2001). 

Analysis of intracellular PHB content of the bacterial cells is an important challenge 

connected with biotechnological production of PHB. The most common technique to 

quantify PHB is based on Gas Chromatography (GC), however, this method does not 

provide rapid tool to analyze PHB content of cells in bioreactor due to labor intensive and 

time-consuming sample preparation. From this point of view, flow cytometry analysis of 

Nile Red stained cells seems to be superior to GC due to its simplicity and rapidity (Vidal- 

Mas et al. 2001). 

The aim of this work was to develop method for analysis of intracellular PHB content 

by using Nile Red staining and Flow Cytometry. Cupriavidus necator H16 (formerly 

Alcaligenes eutrophus, Wautersia eutropha and Ralstonia eutropha) was cultivated in 

5 l bioreactor (Biostat B plus, Sartorius Biotech, Germany) using mineral salt medium 

and waste frying oil as a carbon source (Obruca et al. 2010). Samples were withdrawn at 

regular intervals; cells were pelleted, washed with PBS buffer and fixed with cold ethanol 

(30%, 15 min). After that, the cell suspension (1 ml, approx. 10 6 of cells/ml) was stained 

with 1 ml of Nile Red (1 mg/ml of DMSO). Analysis of cell population was performed 

using Apoggee A50 (Apogee, GB) (excitation at 488 nm, emission was analyzed using 

orange channel). PHB none-producing mutant strain Cupriavidus necator H16/PHB - 4 was 

used as a negative control. 

According to our results, Nile Red staining and Flow Cytometry analysis of PHB cell 

content provides rapid and reliable tool to monitor process of PHB production. The 

interval between sampling and result analysis does not exceed 45 min. which enables to 

control the process of PHB production. 

Furthermore, we developed protocol to stain the bacterial cells without application of 

ethanol fixation. When the cells were stained under slightly elevated temperature (40- 

45°C), the PHA positive cells were stained, but; unlike in case of ethanol fixation, the 

cells remained viable and cultivable. This protocol may be useful for selection of PHAoverproducing 

strains or mutants by Fluorescence Activated Cell Sorting (FACS). 


This work was supported by project “Centre for Materials Research at FCH BUT” No. 

CZ.1.05/2.1.00/01.0012 from ERDF and by the project „Excellent young researcher at 

BUT“ No. CZ.1.07./2.3.00/30.0039. 

References 

Kessler B, Wilholt B (2001) Factors involved in the regulatory of polyhydroxyalkanoate 

metabolism. J Biotech 86: 97–104. 

Vidal-Mas J, Resina-Pelfort O, Haba E, Comas J, Maresa A, Vives-Rego J (2001) Rapid flow 

cytometry – Nile red assessment of PHA cellular content and heterogenitity in cultures 

of Pseudomonas aeruginosa 4ZT2 (NCIB 40044) grown in waste frying oil. Antonie van 

Leeuwenhoek 80: 57-63. 

Obruca, S., Marova, I., Snajdar, O., Mravcova, L. and Svoboda, Z. (2010a) Production of 

poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator from waste rapeseed 

oil using propanol as a precursor of 3-hydroxyvalerate. Biotech Lett 39: 1925-1932. 


P49. NEW ANALOGS OF RHODAMINE 

Jiřina Procházková 1 , Tomáš Pastierik 2 , Peter Šebej 2 , Peter Štacko 2 , Radek Fedr 3 , Petr 

Klán 2 

1 

Dept. of Animal Physiology and Immunology, Institute of Experimental Biology, Faculty 

of Science, Masaryk University, Brno, Czech Republic 

2 

Dept. of Chemistry and RECETOX, Faculty of Science, Masaryk University, Brno, Czech 

Republic 

3 

Dept. of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech 

Republic, v.v.i., Brno, Czech Republic 

Rhodamine derivatives are highly favored in bioimaging and biosensors due to their 

excellent photophysical properties, such as the high molar absorption coefficients, 

excellent fluorescence quantum yields, and good photostability. Two new fluorescent dyes 

have been developed by introducing the ethynyl moiety onto the pyronin chromophore. 

Dyes could be excited by the 630-nm laser and are emitting in far-red region, which is 

extremely advantageous for flow cytometry applications, as the compensation is not 

necessary when measuring the second parameter in green and orange channels. These 

cationic dyes allow quick staining of live cells as the maximal intensity of fluorescence 

within cells in reached in 3 minutes in room temperature. As the Rhodamine dyes 

(e.g., Rhodamine 123 or TMRE) were described to be selectively sequestered in active 

mitochondria and are often used as a markers for mitochondrial membrane potentials 

(MMP), we aimed to analyze not only the photochemical properties of the new dyes but 

also their intracellular localization, ability to function as a sensor of changes in MMP, 

or production of reactive oxygen species, and their affinity to the ABC transporters. We 

confirmed that retention of dyes in dying cells (with decreased MMP) is weaker than in 

live cells. This feature could be thus employed in detection of apoptotic cells, e.g., with 

combination with Annexin V. 


This work was supported by the Grant Agency of the Czech Republic (13-25775S), 

and the project CETOCOEN (CZ.1.05/2.1.00/01.0001) granted by the European 

Regional Development Fund and by grant of Ministry of Education, Youth and Sports 

MSM0021622430. 

References: 

Ward MW, Quantitative Analysis of Membrane Potentials, Live Cell Imaging, Methods in 

Molecular Biology Volume 591, 2010, pp 335-351 


P50. NON-INVASIVE ANALYSIS OF BEATING PARAMETERS IN IN VITRO 

CARDIOMYOCYTE CULTURE 

Dominika Sýkorová 1,3 , Pavel Karas 2 , Jana Navrátilová 1,3 , Lucia Binó 1,3 , Lukáš Kohút 4 , Lukáš 

Kubala 1,3,5 , Jiří Pacherník 1,5 

1 


Republic; 

2 

Centre for Biomedical Image Analysis, Faculty of Informatics Masaryk University, Brno, 


3 

Institute of Biophysics ASCR v.v.i., Brno, Czech Republic; 

4 

Research Center for Toxic Compounds in the Environment, Faculty of Science, Masaryk 

University, Brno, Czech Republic; 

5 


Engineering, St. Anne‘s University Hospital Brno, Czech Republic. 

Studying of tissue and cellular functions via non-invasive methods represents a powerful 

tool for monitoring of various experimental and pathological conditions with minimal 

effect on observed processes. Cardiomyocytes are highly specified cells with unique 

ability of spontaneous beating. The beating characteristics such as beating frequencies 

and chronotropic changes are related to cardiomyocytes differentiation/maturation 

status and are modulated by cell culture conditions. Beating characteristic is modulated 

in response to various stimuli. Response of cardiomyocyte beating to tested compound 

is suggested to be valuable marker for testing cardiotoxicity. However, the detection of 

beating patter of cardiomyocytes in cell culture conditions is highly challenging. 

Here we introduce the Cardio Analyser software developed for digital movie analysis 

of beating properties of cardiomyocytes in vitro culture. Potential and capabilities of 

the software were tested on cardiomyocytes in vitro culture under various experimental 

conditions. Based on our results, this non-invasive method gives promising results in 

studies of the response of cells to various external stimulations, such as differentiation 

promoting agents or treatments. 

Cardio Analyser can be also useful in preliminary drug screening studies as a quick and 

inexpensive tool which doesn’t require extensive expertise. 


This work was supported by grants from MEYS CR MSM0021622430 and Cost CZ 

LD11015, from GAČR 13-29358S, and from the European Social Fund - projects 



CZ.1.05/1.1.00/02.0123). 


P51. FLUORESCENCE-LABELED FERROMAGNETIC NANOPARTICLES FOR MRI-GUIDED 

MAGNETIC FLUID HYPERTHERMIA-BASED GLIOBLASTOMA TREATMENT AND MR 

IMAGING 

Karolina Turnovcova 1 , Vit Herynek 2 , Emil Pollert 3 , Pavel Veverka 3 , Pavel Zvatora 4 , Magda 

Vosmanska 4 , Daniel Jirak 2 , Milan Hajek 2 , Eva Sykova 1 , Pavla Jendelova 1 

1 

Institute of Experimental Medicine ASCR, Prague, Czech Republic, karolina. 

turnovcova@biomed.cas.cz 

2 

MR-Unit, Department of Radiodiagnostic and Interventional Radiology, Institute for 

Clinical and Experimental Medicine, Prague, Czech Republic 

3 

Institute of Physics, ASCR, Prague, Czech Republic 

4 

The Institute of Chemical Technology, Prague, Czech Republic 

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor 

occuring in humans, and its cells are resistant to conventional therapy. Fluorescencelabeled 

ferromagnetic nanoparticles may serve for both diagnostic and therapeutic 

purposes. They represent intracellular labeling using endocytosis uptake, are a suitable 

contrast agent for magnetic resonance imaging (MRI) in vivo, and can also be used for 

guided temperature-controlled thermoablation. The method is based on the deposition 

of stable and nontoxic suspensions of the magnetic nanoparticles inside the tumor 

followed by exposure to a high frequency (HF) electromagnetic field. Magnetic hysteresis 

losses result in local heating by the particles and consequently to the apoptosis of the 

cells. 

Perovskite nanoparticles (La 1-x 

Sr x 

MnO 3 

), coated by SiO 2 

with FITC, were synthesized, their 

surface was modificated with different materials [chitosan, 2-[methoxy(polyethyleneoxy) 

propyl]trimethoxysilane (PEET)], and they were tested in vitro and in vivo. 

Rat and human mesenchymal stromal cells (rMSC, hMSC) and GBM cell lines (C6, A172 

and GaMG) were cultivated with nanoparticles for 48 hours using the xCELLigence System; 

the real-time growth curves were recorded, and the culture viability was analysed by 

trypan blue staining. The nanoparticle uptake into the cells was then analysed using 

FACS-based fluorescence intensity measurements and directly by ICP-MS spectrometry. 

The viability of cells incubated with the nanoparticles was in the range of 60 – 95%, 

whereas a control sample reached 98%. The amount of incorporated nanoparticles was 

up to 10-fold higher in the GBM cell lines and was affected by surface modification. 

Two million C6 cells were injected intradermally into rats (n=10). In 2 weeks, 50 ul of a 

4 mM Mn suspension of perovskite nanoparticles was injected into the glioblastoma 

tissue. MRI images were obtained to confirm the distribution of the nanoparticles in 

the tumor. The rats were exposed to a HF electromagnetic field (480 kHz, 11 mT) for 

30 minutes. Four control animals with tumors underwent HF field exposure without 

nanoparticles, or the application of nanoparticles without exposure to the HF field. The 

temperature in the tumor with nanoparticles increased to 41.7°C, while control animals 

without nanoparticles reached 40°C. Immunohistochemistry (staining for caspase3 

and TUNEL) revealed massive apoptosis in the tumors of the animals with injected 

nanoparticles after exposure to the HF field. 


We can conclude that the targeting of nanoparticles to the appropriate cell type can be 

influenced by specific surface coatings, the distribution of nanoparticles can be easily 

tracked using MRI, and substantial local thermoablation occurs as confirmed by TUNEL 

and caspase3 positivity, which revealed apoptosis in the tumors of animals treated by 

nanoparticles and field exposure. Thus, we were able to confirm that these nanoparticles 

are suitable for MRI-guided thermoablation. 

The study was supported by the grant project No. FR-TI3/521 and by 00023001IKEM. 

P52. TENFOLD EXPANSION OF REGULATORY T CELLS HOMOZYGOUS FOR THE 

CCR5 GENE VARIANT D32 AFTER CD3/CD28 ACTIVATION IN THE PRESENCE OF 

EXOGENOUSLY ADDED RECOMBINANT IL-2 IN VITRO 

Katerina Pavelcova, Julie Chalupnikova, Jan Jencik, Radek Klubal, and Josef Bodor 

Czech Genetic Bank, Prague, Czech Republic; katerina.pavelcova@genetickabanka.cz 

The unprecedented power of the hematopoetic stem cell transplantation of the CCR5 

D32/ D32 cells resistant to HIV has been proved to cure HIV infection in the case of 

leukemic patient (‘Berlin patient’ - Timothy Ray Brown) reported two years ago (Allers 

et al., 2011; Peterson et al., 2013). Successful reconstitution of CD4 + T cells at the 

systemic level as well as in the gut mucosal immune system was found while the patient 

remained without any sign of HIV infection. Furthermore, during the process of immune 

reconstitution, evidence for the replacement of long-lived host tissue cells with donorderived 

cells indicates that the size of the viral reservoir has been reduced over the time. 

Since then another two cases which proved to cure HIV infection after hematopoetic 

stem cell transplantation of the CCR5 D32/D32 cells were reported from Brigham and 

Women Hospital in Boston (McNutt, 2013). 

Viral entry into CD4 + T cells is mediated by the interaction with a cellular chemokine 

receptor, the most common of which are CCR5 and CXCR4 (Allers et al., 2011). The 

major barrier to viral eradication in patients receiving antiretroviral therapy (ART) is the 

establishment of HIV reservoirs. Cells of persons homozygous for the CCR5 gene variant 

D32 (CCR5D32/D32) are naturally resistant to infection with CCR5-tropic HIV strains 

(R5 HIV) because of the lack of functional CCR5 cell-surface expression (Peterson et al., 

2013). Our data based on the general population in Czech Republic show a frequency 

of approximately 20% heterozygous persons. Four homozygous persons bearing D32 

mutation (CCR5D32/D32) were identified out of 709 individuals tested based on the 

genotyping of CCR5 D32 deletion. 

Due to their far-ranging tolerizing capability regulatory T cells (Tregs) have become key 

targets in the development of tolerance-inducing therapies (Wing et al., 2010). Like 

other T cells, Tregs require activation for their expansion and/or function. Moreover, 

we evaluated the frequency of Tregs in persons homozygous for the CCR5 gene variant 

D32 (CCR5D32/D32) compared with healthy control patients (wt) to make sure that the 

potential amelioration of GvHD after HSC transplantation of the cells resistant to HIV 


could be achieved via the activation of Treg‘s suppressive function. 

Here we report a more than tenfold increase in the frequency of Tregs following CD3/ 

CD28 co-stimulation within a week of in vitro cultivation of human Tregs, irrespective 

of their genotype. Somewhat decreased kinetics of Treg expansion were observed on 

day 5 in CD4 + T cells in the person homozygous for the CCR5 gene variant D32 (21.5% 

versus 74.7% in wt) (Fig. 1). Importantly, similar the treatments, which lead to the 

activation of Treg function in humans – e.g. anti-CD3/CD2/CD28 stimulation (Hagness 

et al. 2012) simultaneously drove expansion of Tregs e.g. using anti-CD3/CD28 and/ 

or IL-2 (see Fig.1). Our study demonstrates a useful tool for in vitro evaluation of Treg 

function and facilitates further understanding of the mechanisms of immunological selftolerance, 

which may also provide insights into how strong immune responses, such as 

graft rejection, can be restrained and engraftment of HIV resistant cells in HIV patients 

with AIDS lymphoma or leukemia can be augmented. 

References 

Allers, K., Hütter, G., Hofmann, J., Loddenkemper, C., Rieger, K., Thiel, E., and Schneider, 

T.: Evidence for the cure of HIV infection by CCR5 D32/ D32 stem cell transplantation. - 

Blood 117: 2791-2799, 2011. 

Hagness, M., et al.: Kinetics and activation requirements of contact-dependent immune 

suppression by human regulatory T cells. – The Journal of Immunology 188: 5459-5466, 

2012. 

McNutt, M.: News of the Week, Evidence mounts for two more HIV cures. Science 341: 

114, 2013. 

Peterson, C.W., Younan, P., Jerome, K.R., and Kiem, H.P.: Combinatorial anti-HIV gene 

therapy: using a multipronged approach to reach beyond HAART. Gene Therapy 20: 695- 

702, 2013. 

Wing, K., Sakaguchi, S.: Regulatory T cells exert checks and balances on self tolerance 

and autoimmunity. Nature Immunology 11: 7-13, 2010. 

Caption to figure 

Fig. 1. Tregs homozygous for the CCR5 gene variant 

D32 were expanded in vitro tenfold after CD3/ 

CD28 activation in the presence of exogenously 

added recombinant IL-2. CD4 + T-cell expression 

of the activation marker CD25 and lack of CD127 

expression was evaluated by flow cytometry in 

cells homozygous in the CCR5 gene variant D32 

(CCR5 D32/ D32) or wt (wt/wt) and activated in 

vitro by CD3/CD28 mAbs coated on plastic in the 

presence of exogenously added IL-2. In vitro expansion of Tregs lead to an approximately 

tenfold enrichment, irrespective of genotype, under the conditions mentioned above as 

monitored after seven days of cultivation. 


P53. SELECTED UNQUESTIONABLE ONCOLOGIC DIAGNOSIS IN CASE OF FLOW 

CYTOMETRY APPLICATION 

Jana Chovancová 1,2 , Olga Stehlíková 1 , Tomáš Bernard 1 , Jiří Mayer 1,2 , Michael Doubek 1,2 

1 

Department of Internal Medicine – Hematology and Oncology, University Hospital, Brno 

and Faculty of Medicine, Masaryk University, Brno, Czech Republic 

2 

Central European Institute of Technology, Masaryk University, Brno, Czech Republic 

The aim of our study is to show some hematological diagnosis as well as nonhematological 

disorders in different stages, which were identified in our laboratory 

of flow cytometry and could cause confusion from the flowcytometric point of view. 

Following disorders have been selected: T-cell prolymphocytic leukemia (T-PLL), acute B 

lymphoblastic leukemia (B-ALL), where the presence of pathologic cells is extremely low, 

and bone marrow metastatic infiltration of rhabdomyosarcoma (RMS). 

T-PLL is a rare, aggressive lymphoid malignancy with characteristic morphologic, 

immunophenotypic, cytogenetic, and molecular features. However, the flow cytometric 

discrimination of T-PLL from other T-cell disorders might be challenging. The case is 

presented of 65-year-old man, showing the pathologic population with hyperexpression 

of antigens CD5 and CD7, negative expression of cytoplasmatic TdT, surface antigen 

CD3 and TCR. Thus, the whole measured phenotype is CD2 + s3 - c3 + 4 + 5 ++ 7 ++ 8 - cTdT - TCR - 

(Dearden, 2012). 

B-ALL is quite common leukemia in children, so detecting usually does not cause any 

trouble. What could lead to make misdiagnosis is a type of relapse which presents just a 

low level of blastic cells in peripheral blood (< 1 % of leukocytes) that is hardly detected 

by morphology approach. Since a pathologic population is commonly CD45 negative, the 

small number of blastic cells hides in the area of erythrocytes, trombocytes and debris 

showing fallacious healthy results. 

RMS is the most common soft tissue sarcoma in children under 15 years. The patients 

typically present with a tumor mass in the head and neck region, urogenital tract or lower 

extremities. Unusual massive bone marrow infiltration that could imitate hematologic 

neoplasm is presented of a teenage boy, where typical phenotype CD45 - 56 ++ 57 + 90 + of 

pathologic cells was found (Bozzi et al., 2008). 

Flow cytometry is a well-established approach for diagnosing hematological and 

lymphoid disease as well as detecting metastatic cells of some solid tumors in bone 

marrow. Nevertheless, considering some rare cases results could lead to deceptive 

conclusions. 


This work was supported by grant MUNI/A/0723/2012. 

References 

Bozzi F, Collini P, Aiello A, Barzann E, Gambirasio F, Podda M, Meazza C, Ferrari A, Luksch 

R.: Flow Cytometric Phenotype of Rhabdomyosarcoma Bone Marrow Metastatic Cells 

and its Implication in Differential Diagnosis with Neuroblastoma – Anticancer Research 


28. Pp. 1565-1570. 2008. 

Dearden C: B- and T-cell Prolymphocytic Leukemia: Antibody Approaches - Hematology 

Am Soc Hematol Educ Program. Pp. 645-651. 2012. 

P54. PHENOTYPE ANALYSIS OF DIFFERENTIATION: FROM HEMATOPOIETIC STEM 

CELLS TOWARD B CELLS IN MONOCLONAL GAMMOPATHIES 

Renata Bezděková 1 , Lucie Říhová 1,2 , Pavla Všianská 1,2,3 , Tamara Varmužová 1,2 , Zdenka 

Pavková 1,2 , Renata Suská 2 , Miroslav Penka 2 , Roman Hájek 1,2,4 

1 

Babak Myeloma Group by Department of Pathological Physiology, Faculty of Medicine, 

Masaryk University, Brno, Czech Republic; Renatta.Bezdekova@seznam.cz 

2 

Department of Clinical Haematology, University Hospital Brno, Brno, Czech Republic 

3 



4 

Department of Clinical Hematology, University Hospital and Faculty of Medicine, 

Ostrava University, Ostrava, Czech Republic 

Background: Monoclonal gammopathies (MG) are characterized by presence of clonal 

plasma cells (PC) producing monoclonal immunoglobulin (MIG). Non-malignant 

monoclonal gammopathy of undetermined significance (MGUS) could progress 

into malignant multiple myeloma (MM) (Pérez-Persona et al., 2007). The cause of 

pathological transformation in MGs is still not known. Probably, there is a defect at the 

level of memory B cells from which the PCs are subsequently developed (Matsui et al., 

2008). However, the early and immature form of B lymphocytes could be also involved. 

The existence of myeloma-initiating cells is taken into consideration. These cells have 

characteristic properties of stem cells and could be responsible for eventual relapse of 

MM after treatment (Hosen, 2013). 

Aim: Flow cytometry analysis and enumeration of different stem cell and B cell 

subpopulations in monoclonal gammopathy subjects. 

Subjects and methods: There were analysed 22 MM and 15 MGUS untreated subjects. 

Whole bone marrow was incubated with followed antibodies (CD38, CD45, Lin, CD133, 

CD34, CD117, CD243, CD138, HLA-DR, CD43, CD24, CD19, CD10, and CD20) and after 

lysis analysed by flow cytometer. Different subpopulations were detected according to 

their phenotype profile. 

Results: There was found no difference in total number of CD34 + stem cells (including 

early progenitors) when compared MGUS and MM. Number of CD117 - CD133 - lymphoid 

progenitors from CD34 + stem cells was statistically significantly increased in MGUS than 

in MM (14.5 vs. 8.5 %; p

significance (5.3 vs. 2.6 %). On the other hand, percentage of mature B lymphocytes was 

significantly decreased in MGUS when compared with MM (62.7 vs. 83.8 %; p

cells (SFC) using IFN-γ Elispot in comparison to controls. Moreover, a trend to higher 

numbers of SFC was observed in these patients when compared to persons with a low 

frequency of recurrences (n=7). Additionally, lack of difference was found in CD38 and 

HLA-DR expression on circulating CD8+ T cells between the study groups. 

Overall, these results indicate that systemic HSV-specific T cells are not suppressed 

during recurrent genital herpes and the increased frequencies of these cells in the blood 

are associated with the disease severity rather than with a protective role of these cells. 


This work was supported by the grant of Charles University in Prague, PRVOUK/P24/LF1/3. 

References 

Wald A, Zeh J, Selke S, et al. Reactivation of genital herpes simplex virus type 2 infection 

in asymptomatic seropositive persons. N Engl J Med 2000;342:844-50. 

Zhu J, Koelle DM, Cao J, et al. Virus-specific CD8+ T cells accumulate near sensory nerve 

endings in genital skin during subclinical HSV-2 reactivation. J Exp Med 2007;204:595-603. 

P56. REDUCED ALLERGY INCIDENCE AND INCREASED ACTIVITY OF TREGS IN SIX YEAR 

OLD CHILDREN OF ALLERGIC MOTHERS COLONIZED AT BIRTH BY PROBIOTIC E. COLI 

Jiří Hrdý 1 , Ingrid Kocourková 2 , Rája Lodinová-Žádníková 2 , Ludmila Prokešová 1 

1 

Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University 

in Prague, Prague, Czech Republic; jiri.hrdy@lf1.cuni.cz 

2 

Institute for the Care of Mother and Child, Prague, Czech Republic 

Allergy belongs to one of the most common diseases with still increasing incidence. 

In our previous work, we reported decreased allergy incidence in probiotic (Colinfant 

New Born, Escherichia coli O83:K24:H31) colonized children [Lodinová-Žádníková, 2003, 

Lodinová-Žádníková, 2010]. 

In this work, significantly decreased allergy incidence in colonized children of allergic 

mothers (children with high risk for allergy development) in comparison to noncolonized 

children of allergic mothers was observed. Allergy incidence in colonized 

children of allergic mothers was comparable with allergy incidence in non-colonized 

children of healthy mothers (children with relatively low risk for allergy development). 

The beneficial effect of probiotics is well acknowledged but the mechanisms of their 

actions are still unclear. 

Because regulatory T cells (Tregs) are known to set the tolerance to relatively innocuous 

environmental antigens (allergens), the proportion and functional properties of Tregs 

from peripheral blood of colonized and non-colonized children in relation to their allergy 

status were followed by flow cytometry. Surface markers of Tregs, intracellular FoxP3 

and regulatory cytokines IL-10 and TGF-beta were tested. 

The Treg percentage was lower in the blood of healthy children but the MFI (median of 

fluorescence intensity) of FoxP3 reflecting better the functional potency was higher in 

Tregs of healthy children in comparison with allergic ones. Furthermore, intracellular 

presence of IL-10 was detected in higher percentage of Tregs of healthy children in 


comparison to allergic children. The differences in intracellular TGF-beta were not 

significant but the tendency to higher expression of TGF-beta in Tregs of healthy children 

was obvious. Colonized children, even these suffering from allergy, showed better 

functional markers of Tregs when compared with cells of non-colonized allergic children. 

It is possible to conclude the insufficient function of Tregs in allergic children leads to 

compensatory increase of their number. The immunomodulatory effect of probiotic E. 

coli can be, at least partially, explained by its positive influence on Treg function. 


This work was supported by Charles University research project PRVOUK P25/LF1/2, 

GAUK259911, MSM0021620806. 

References 

Lodinová-Žádníková, R., Cukrowská, B., Tlaskalová-Hogenová, H.: Oral administration 

of probiotic Escherichia coli after birth reduces frequency of allergies and repeated 

infections later in life (after 10 and 20 years). Int Arch Allergy Immunol 2003; 131: 209– 

211. 

Lodinová-Zádníková, R., Prokesová, L., Kocourková, I., Hrdý, J., Zizka, J.: Prevention of 

allergy in infants of allergic mothers by probiotic Escherichia coli. J. Int Arch Allergy 

Immunol. 2010;153(2):201-6 

P57. SIGNIFICANT ANTIGENS FOR DETECTION OF MINIMAL RESIDUAL DISEASE IN 

PATIENTS WITH MANTLE CELL LYMPHOMA USING FLOW CYTOMETRY APPROACH 

Jana Chovancová 1,2 , Tomáš Bernard 3 , David Šálek 3 , Andrea Janíková 3 , Jiří Mayer 1,2,3 , 

Michael Doubek 1,2,3 

1 

Faculty of Medicine Masaryk University, Kamenice 5, 625 000 Brno, Czech Republic; 

jchovan@mail.muni.cz 

2 

Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, 


3 

Dept. of Internal Medicine – Hematology and Oncology, University Hospital, Jihlavska 

20, 625 00 Brno, Czech Republic 

Background: Mantle cell lymphoma (MCL) is a B-cell neoplasm with an aggressive 

clinical course typically characterized by CD5 + 19 + pathologic lymphocytes. The increase 

of efficient treatment options has brought needs for early detection of complete 

remission and minimal residual disease (MRD) (Medd et al., 2011). In this study, we 

propose suitable combinations of CD markers that could lead to effective detection of 

MRD in order to increase its sensitivity which means to improve the prediction of a 

therapy response or continued remission. 

Methods: Cohort of 28 consecutive patients with a new diagnosis of MCL together with 

16 healthy donors were included into the analysis. Samples of peripheral blood were 

tested using eight-color flow cytometry protocol. Cell surface antigens were stained with 

fluorescence labeled monoclonal antibodies (anti-CD5, 10, 19, 20, 21, 22, 23, 24, 35, 

38, 43, 45, 62L, 79b, 196 and 200). Flow cytometric acquisition was performed on a 


FACSCantoII flow cytometer (Becton Dickinson, NJ, USA). 

Results: There were found significantly lower expression of antigens CD23, CD62L, CD196 

and CD200 on MCL B lymphocytes compared to healthy controls (p

(EXBIO, clone MEM-63). The immunophenotype of the leukemic cells were identified at 

diagnosis by 5-color panels. The MRD detection at day 15 was carried out according to 

the ALL IC-BFM 2009 flow cytometric MRD protocol. In the control bone marrows the 

precursor B-cells were gated by their CD19, CD45, CD10, FSC and SSC parameters. 

Using the median CD58 MEFL values of the leukemic population the 3 groups of samples 

were compared by Mann-Whitney U test and ROC analysis was performed. 

In agreement with previous studies we found significant difference (in average 6.3 times 

overexpression) between diagnostic samples (mean MEFL= 7109) and control bone 

marrows (mean MEFL=1127). However in the day 15 samples we found a decrease 

compared to the diagnostic values in most (16 of 17) cases (mean MEFL=3494). The 

relative intensity decreased in 11 of 17 cases below 50% of the original value. We tried 

to define cut-off values to separate CD58 overexpressed samples. The best cut-off value 

for the day 15 samples was 1367 but with significantly lower specificity (81.8%) and 

sensitivity (70.6%) compared to the excellent cut-off value (MEFL=2741, specificity: 100% 

and sensitivity: 95.2%) of the diagnostic samples. When applying the higher original cutoff 

to the day 15 samples the results show no overexpression of CD58 in more than half 

of the patients. 

In contrast with previous studies we found a remarkable decrease in the CD58 expression 

during the induction phase of the ALL IC-BFM 2009 treatment protocol at day 15. In conclusion, 

due to its downregulation, CD58 should be treated with caution as an MRD marker to 

avoid underestimation of residual leukemic cells in day 15 p-B-ALL bone marrow samples. 

References 

Jiann-Shiuh Chen, Elaine Coustan-Smith, Toshio Suzuki, Geoffrey A. Neale, Keichiro 

Mihara, Ching-Hon Pui, and Dario Campana.: Identification of novel markers for 

monitoring minimal residual disease in acute lymphoblastic leukemia. Blood, 97: 2015- 

2020, 2001. 

Veltroni M, De Zen L, Sanzari MC, Maglia O, Dworzak MN, Ratei R, Biondi A, Basso G, 

Gaipa G; I-BFM-ALL-FCM-MRD-Study Group. Expression of CD58 in normal, regenerating 

and leukemic bone marrow B cells: implications for the detection of minimal residual 

disease in acute lymphocytic leukemia. Haematologica, 88(11):1245-52, 2003. 

Gaipa G, Basso G, Maglia O, Leoni V, Faini A, Cazzaniga G, Bugarin C, Veltroni M, Michelotto 

B, Ratei R, Coliva T, Valsecchi MG, Biondi A, Dworzak MN; I-BFM-ALL-FCM-MRD Study 

Group. Drug-induced immunophenotypic modulation in childhood ALL: implications for 

minimal residual disease detection. Leukemia,19(1):49-56, 2005. 

Lee RV, Braylan RC, Rimsza LM.: CD58 expression decreases as nonmalignant B cells 

mature in bone marrow and is frequently overexpressed in adult and pediatric precursor 

B-cell acute lymphoblastic leukemia. Am J Clin Pathol. 123(1):119-24, 2005. 

Coustan-Smith E, Song G, Clark C, Key L, Liu P, Mehrpooya M, Stow P, Su X, Shurtleff S, 

Pui CH, Downing JR, Campana D. New markers for minimal residual disease detection in 

acute lymphoblastic leukemia. Blood, 117(23):6267-76, 2011. 


P59. EARLY DIAGNOSTICS OF CARTILAGE-HAIR HYPOPLASIA AS A CAUSE OF SEVERE 

COMBINED IMMUNODEFICIENCY (SCID) USING FLOW CYTOMETRY AND TREC/KREC 

ANALYSIS 

Kotrová Michaela 1 , Formánková Renata 1 , Mejstříková Ester 1 , Froňková Eva 1 , Říha Petr 1 , 

Svatoň Michael 1 , Baxová Alice 2 , Šedivá Anna 3 , Sedláček Petr 1 , Starý Jan 1 

1 

Department of Paediatric Haematology and Oncology, 2 nd Faculty of Medicine, Charles 

University in Prague and Motol University Hospital michaela.kotrova@lfmotol.cuni.cz 

2 

Institute of Biology and Department of Medical Genetics 1 st Faculty of Medicine, 

Charles University, Prague and General Teaching Hospital 

3 

Department of Immunology, 2 nd Faculty of Medicine, Charles University in Prague 

and Motol University Hospital 

Cartilage-hair hypoplasia (CHH), also known as metaphyseal chondrodysplasia, is a rare 

autosomal recessive genetic disorder caused by mutations of the RMRP gene, encoding 

non-protein RNA Component of Mitochondrial RNA Processing Endoribonuclease. 

Mutations in this area can lead to a wide spectrum of signs and symptoms including 

different degrees of short stature, hair hypoplasia, defective erythrogenesis, and 

immunodeficiency. The immunodeficiency in CHH can present as isolated T-cell or B-cell 

immunodeficiency, or combined T-cell and B-cell immunodeficiency. In CHH patients, 

allogeneic hematopoietic stem cell transplantation (HSCT) can correct immune deficiency 

and anemia but has no effect on skeletal changes. 

A 2.5 months girl presented with systemic cytomegalovirus (CMV) disease, CMV 

interstitial pneumonia and rotaviral enteritis. Flow cytometry evaluation of peripheral 

blood revealed 67% of granulocytes, 6.2% of lymfocytes and 23% of monocytes according 

to CD45+14++. CD19+ B lymphocytes prevailed, the T lymphocytes were in minority 

(4.6%) and only CD3+4+ and TCR gamma/delta cells were present. Within CD3+4+cells, 

98.2% of cells were CD45RO positive, suggesting maternofetal engraftment or oligoclonal 

expansion of residual T cells. B cell compartment consisted mostly of IgD+CD27neg naïve 

B cells (97%), IgMnegIgDnegCD27pos class switched memory B cells and plasmablasts 

were almost missing (0.18%). The diagnosis of SCID was subsequently confirmed by 

recombination circle quantitative PCR analysis aiming at detection of newly derived T 

(„TREC“) and B („KREC“) cells. TRECs were not present in the peripheral blood, while 

KREC levels did not differ from those in healthy newborns, indicating complete early 

block in alpha/beta T-cell development and normal development of B lymphocytes until 

the stage of class switching. The patient has successfully undergone HSCT at the age of 

three months with unrelated matched cord blood. 

The exome sequencing was performed with the aim to find underlying mutation after the 

exclusion of IL7R defect. However, neither any mutation in ~20 known genes associated 

with T-B+ SCID, nor any new, previously undescribed mutation was found. Skeletal 

dysplasia was detected in the patient by ultrasound already in the 36th gestational 

week and diagnosis of CHH was suggested by clinical genetician at the age of 5 months. 

As non-protein coding gene, RMRP was not covered sufficiently in exome sequencing 

library (Nimblegen SeqCap EZ Human Exome Library v2.0). We performed a molecular 


genetic analysis of RMRP gene based on classical sequencing which revealed that the 

girl is a compound heterozygote for mutations affecting the RMRP gene. She inherited 

paternal duplication in promoter region causing transcription impairment; on maternal 

allele we detected a point mutation at position 35 of the gene. This position is strongly 

conserved in the entire class of mammals. 

Now, 15 months after HSCT, our patient is alive and well. 

Conclusion: We present the youngest patient so far transplanted for cartillage hair 

hypoplasia due to severe immunodeficiency. The diagnostic pipeline for primary 

immunodeficiencies was extended to TREC/KREC analysis, enabling robust assessment 

of the number of newly emerging lymphocytes. Although not leading to definitive 

diagnosis in this particular case, exome sequencing seems to be relatively cheap and fast 

variant to classical sequencing of individual genes. 


Supported by the project for conceptual development of research organization 00064203. 

E.F. was supported by the L‘Oreal-UNESCO program “For Women in Science” 2013. 

References 

Bonafé L, Dermitzakis ET, Unger S, Greenberg CR, Campos-Xavier BA, Zankl A, Ucla C, 

Antonarakis SE, Superti-Furga A, Reymond A: Evolutionary comparison provides evidence 

for pathogenicity of RMRP mutations. PLoS Genet. 2005 Oct;1(4):e47. 

Bordon V, Gennery AR, Slatter MA, Vandecruys E, Laureys G, Veys P, Qasim W, Friedrich W, 

Wulfraat NM, Scherer F, Cant AJ, Fischer A, Cavazzana-Calvo M,Bredius RG, Notarangelo 

LD, Mazzolari E, Neven B, Güngör T; Inborn Error Working Party of the European Bone 

Marrow Transplantation (EBMT) group: Clinical and immunologic outcome of patients 

with cartilage hair hypoplasia after hematopoietic stem cell transplantation. Blood. 2010 

Jul 8;116(1):27-35. 

Thiel CT, Mortier G, Kaitila I, Reis A, Rauch A: Type and level of RMRP functional 

impairment predicts phenotype in the cartilage hair hypoplasia-anauxetic dysplasia 

spectrum. Am J Hum Genet. 2007 Sep;81(3):519-29. 

P60. THE SPECIFICITY OF CYTOMETRIC DETECTION OF HLA-B27 IS COMPARABLE TO 

MICROCYTOTOXIC TEST 

Olga Kryštůfková, Klára Prajzlerová, Hana Hulejová, Ivana Půtová 

Department of Clinical Immunology of the Institute of Rheumatology, Prague, Czech 

Republic and Dept. of Rheumatology, 1 st Faculty of Medicine, Charles University, 

Prague, Czech Republic, krys@revma.cz 

A strong association between HLA-B27 and ankylosing spondylitis or other diseases 

from a group of axial spondyloarthritis (SpA) has been demonstrated. HLAB-27 positivity 

has been included into SpondyloArthritis international Society classification criteria 

for axial SpA (Rudwaleit, 2009). HLA-typing performed with the microcytotoxic tests 

is time consuming and expensive if used for HLA-B27 screening, therefore alternative 

flow cytometry method was developed (Pei, 1993). HLA-B27 belongs to the HLA-B7 


cross-reacting group (CREG) and share common epitopes with HLA-B7. In addition, 

cross-relativities of HLA-B27 typing reagents (including IVD monoclonal antibodies) with 

non-HLA-B7 CREG antigens, have been reported (Levering, 2003). 

According to the European Federation for Immunogenetics guidelines, combinations 

of two different anti-HLA-B27 clones should be used to optimally control for crossreactivity. 

Therefore we included FITC-labelled monoclonal antibodies of three clones 

into the routine diagnostics. Clone ABC-m3 (IOTest HLA-B27-FITC/HLA-B7-PE; Beckmann 

Coulter) was used for screening and for confirmation in HLA-B7 positive samples; 

GS145.2 (HLA-B27 Kit; Becton Dickinson Biosciences) and FD705 (HLA-B27-FITC; One 

Lambda ) were included. 

Because the manufacturer of IOTest, recommends adjustment of the cytometer on blood 

with fully documented HLA phenotype, the comparative results from microcytotoxic 

assay (HistoTray B27; BAG Health Care GmbH) of 117 consecutive patient samples 

were used. After daily standard set up of cytometer CyAN ADP TM with calibration 

beads (SPHERO TM Rainbow Calibration Particles, Spherotech), the median fluorescence 

intensity (MFI) was recorded. Cut off value of HLA-B27 positivity was calculated from 

HLA-B27 negative samples as the mean + 4 s.d. in 116 samples with 100% sensitivity but 

only 91% specificity. With exclusion of HLA-B7 positive samples (27%), and detection of 

cut off with use of ROC analysis, the cytometric assay has shown comparable results to 

the microcytotoxic assay with 100% specificity. Confirmatory analysis of HLA-B7 positive 

samples with GS145.2 (set up on HLA-B27 kit calibration beads) and FD705 (cut off = 

mean + 4 s.d.), available in 17 samples, shown 100% sensitivity and 100% specificity. 

However, from total 362 consecutive routine samples analysed within following three 

months; 16 samples (4%) shown discordant results of staining with upper mentioned 

three clones and were recommended for reanalysis with other method. We cannot 

exclude cross reactivity with other HLA-B7 CREG and non-HLA-B7 CREG antigens as was 

shown by Levering et al. 


Institutional support of MZČR 0002372801 

References 

Rudwaleit, M., D. van der Heijde, et al. (2009). “The development of Assessment of 

SpondyloArthritis international Society classification criteria for axial spondyloarthritis 

(part II): validation and final selection.” Ann Rheum Dis 68(6): 777-783. 

Pei, R., M. Arjomand-Shamsai, et al. (1993). “A monospecific HLA-B27 fluorescein 

isothiocyanate-conjugated monoclonal antibody for rapid, simple and accurate HLA-B27 

typing.” Tissue Antigens 41(4): 200-203. 

Levering, W. H., H. Wind, et al. (2003). “Flow cytometric HLA-B27 screening: crossreactivity 

patterns of commercially available anti-HLA-B27 monoclonal antibodies with 

other HLA-B antigens.” Cytometry B Clin Cytom 54(1): 28-38. 


P61. PARAMETERS OF CELLULAR IMMUNITY IN PATIENTS WITH RHEUMATIC DISEASES 

DURING PREGNANCY 

Gabriela Marková 1 , Jana Špajdelová 1 , Stanislava Blažíčková 1,2 

1 

Faculty of Health Care and Social Work, University of Trnava, Trnava, Slovak Republic; 

gabimarkova@gmail.com 

2 

Laboratoria s.r.o., Piešťany, Slovak Republic 

Rheumatoid arthritis and systemic lupus erythematosus have in part overlapping clinical 

features, but behave strikingly differently during pregnancy. Pregnancy patients with SLE 

have a double incidence of flare, whereas diminution of disease activity in observed 

in patients with RA. Apparently, an immunomodulating mechanism is activated during 

pregnancy that is responsible for opposite effect in activity of these autoimmune 

diseases. Both antibody and cellular immunities are maintained. Since the immune 

mechanisms plays a role in the pathogenesis of rheumatic diseases, we monitored 

the changes in selected parameters of cellular immunity in 5 pregnant patients with 

rheumatic diseases (2 SLE patients, 2 PsA patients and 1 JCA patient) in the 6 th and 9 th 

months of pregnancy, a day after delivery and 2 months after delivery. The evaluated 

parameters were: subpopulations of leucocytes in peripheral blood and their activity. 

In the course of pregnancy, slight increase of T-lymphocytes, especially of helper CD4 

lymphocytes was observed, as well as decrease of cytotoxic CD8 lymphocytes. After 

childbirth the values of all these parameters went back to the standard. There were no 

alterations observed in the numbers of NK cells either in the course of pregnancy or after 

delivery. The expression of the CD69 and CD25 receptors on the lymphocytes stimulated 

with phytohemaglutinin and CD2/CD2R monoclonal antibody decreased in pregnancy. 

A possibility that, observed alterations in the cellular and non-specific immunities can 

contribute in the immune homeostasis disorder in rheumatic patients after delivery, 

cannot be excluded. 

P62. DETERMINATION OF REGULATORY T CELLS IN PATIENTS WITH SELECTIVE IGA 

DEFICIENCY 

Jana Nechvatalova, Jiri Litzman, Marcela Vlkova 

Department of Clinical Immunology and Allergology, St Anne’s University Hospital, 

Faculty of Medicine, Masaryk University, Brno, Czech Republic; jana.nechvatalova@ 

fnusa.cz 

Previous studies showed that several T- or B-lymphocyte abnormalities seen in the most 

frequent symptomatic immunoglobulin deficiency, common variable immunodeficiency 

(CVID), were also observed in a genetically related state - selective IgA deficiency (IgAD) 

(Litzman et al., 2007, Nechvatalova et al., 2012). Patients with IgAD are asymptomatic 

in most cases, or they may suffer from recurrent infections of the respiratory tract; also 


the frequency of autoimmune diseases is increased in IgAD. In this study we focused on 

determination of regulatory T cells in these immunodeficient patients. 

Peripheral blood was obtained from 80 patients with IgAD, 48 patients with CVID and 80 

healthy controls. Phenotyping was performed by flow cytometric analysis. T reg 

cells were 

defined as CD4 + CD25 high CD127 low . The autoantibodies determined included rheumatoid 

factor, antinuclear, anti-thyroid peroxidase, anti-thyroglobulin, anti-gastric parietal cells, 

anti-smooth muscle, and anti-tissue transglutaminase antibodies. Statistical analysis was 

performed by the Mann-Whitney test. 

We documented a decrease of T reg 

cells in patients with CVID compared to controls 

(P

P63. THEORETICAL AND PRACTICAL ASPECTS OF QUANTITATIVE FLUORESCENCE 

CYTOMETRY 

Michaela Pevná, Martin Klabusay 

International Clinical Research Center – Integrated Cellular Center Therapy and 

Regenerative Medicine, St. Anne’s University Hospital Brno, Brno, Czech Republic; 

michaela.pevna@fnusa.cz 

The aim of the quantitative fluorescence cytometry (QFCM) method is to determine the 

exact amount of the specific antigen on the cell surface. The QFCM theory is based on 

the estimated correlation between the amount of antigen and fluorescence intensity 

(FI) of fluorochrome conjugated monoclonal antibody bound to the antigen. Due to the 

diversity of instruments, their settings and output variability of the device, for antigen 

quantification we use calibration particles with a known amount of fluorochrome or 

epitopes. However, QFCM in reality brings numerous technical problems and despite two 

decades of its existence, the method has not been standardized at an inter-laboratory 

level yet. 

Fluorescence is only one of the deactivation processes, occurring after the excitation 

of an electron that is induced by photon absorption in chromophores. Fluorescence 

efficiency is best expressed as the quantum yield of fluorescence Φ f 

. Its value is reduced 

by competing deactivation processes: phosphorescence, radiationless deactivation, 

photochemical reactions and quenching. FI is equal to the product of the light absorbed 

(derived from the Lambert-Beer law) and quantum yield Φ f 

. If we compare the FI of 

an unknown sample and calibration particles, the fluorophore microenvironment (e.g. 

solvent, pH and temperature) plays a crucial role and the values of molar extinction 

coefficients ε, absorption and emission spectra (and ideally also quantum yields Φ f 

) in 

both cases should be identical (Klán, 2001). 

The fluorescent signal is produced in the flow chamber, where the flow system provides 

accurate passage of individual cells through the center of the laser beam. Due to the speed 

deviations and the change from the central position, the cells are exposed for different 

times to different laser intensities. Then the collecting lens transmits only the light 

emitted into a solid angle (not from the whole cell).The transmission of the transmitted 

light by interference filters does not reach 100 % and their spatial configuration varies 

among devices. The characteristics of photomultiplier tubes (sensitivity, quantum 

efficiency and variable gain) and radiant flux P of incident light determine the size of 

the output current. The signal in the form of voltage (coming from the preamplifier) is 

processed by the peak detectors. Whether the peak height or the peak area is suitable 

for QFCM, it depends on the ratio of cell size and diameter of the laser beam. The system 

linearity, requirement for accurate QFCM measurement, strongly depends mainly on the 

proper operation of amplifiers. Noise, background fluorescence and autofluorescence 

limits the possibility of weak fluorescence signal detection (in the first decade on the 

logarithmic scale). Adequate spectral compensation settings are necessary for QFCM 

measurements (Shapiro, 2004). 

The binding of the antigen (Ag) with the monoclonal antibody (mAb) is mediated by 


non-covalent bonds. For a solution of Ag and mAb, it is valid that under the conditions 

of chemical equilibrium, the ratio of the concentration of the complex and the product 

of the concentrations of the reactants are equal to the affinity, which is a constant at 

given temperature, pH and salt concentration. For the sample with cells, we always look 

for saturating concentrations of mAb for the target antigen. The number of cells and the 

reaction volume is critical, which dramatically alters the default concentration values for 

Ag and mAb. The mAb binding to a specific antigen may be monovalent or bivalent, or 

may be protected by spatial effects. High concentrations of mAb promote non-specific 

binding with low affinity (Berzofsky, 1999). 

QFCM results are usually presented in MESF (molecules of equivalent soluble 

fluorochrome) or ABC (antibody binding capacity) units. While the values in MESF units 

are affected by the fluorochrome/protein ratio of different lots of antibodies, for interlaboratory 

comparison ABC units with known amount of epitopes are appropriate. 

QFCM can determine the level of target antigen expression, for instance on tumor cells 

of patients undergoing the treatment with monoclonal antibody. This fact could have 

important consequences for pharmacokinetics of such a treatment. Therefore QFCM 

should become more widely recognized method in clinical flow cytometry, especially in 

oncology. 


This work was supported by ERDF-FNUSA-ICRC (No.CZ.1.05/1.1.00/02.0123). 

References 

Berzofsky, J. A., Berkower, I. J., Epstein, S. L.: Antigen-Antibody Interactions and 

Monoclonal Antibodies. - In: Paul W.E. (ed.) Fundamental Immunology, Fourth Edition. 

Pp. 75-77. Lippincott-Raven, Philadelphia, 1999. 

Klán, P.: Absorpce a emise záření. – In: P. Klán (ed.) Organická fotochemie. Pp.15-30. 

Masarykova univerzita, Brno, 2001. 

Shapiro, H. M.: How flow cytometers work. – In: H. M. Shapiro (ed.) Practical Flow 

Cytometry, 4th Edition. Pp. 101-223. Wiley Liss, New York, 2004 

P64. MONOCLONAL GAMMOPATHY ANALYSES USING EUROFLOW APPROACH 

Lucie Rihova 1,2 , Pavla Vsianska 1,2 , Tamara Varmužová 1,2 , Zdenka Pavkova 1,2 , Renata 

Suska 1 , Renata Bezdekova 2 , Miroslav Penka 1 , Roman Hajek 1,2,3 

1 

Department of Clinical Haematology, University Hospital Brno, Brno, Czech Republic; 

lucie.rihova@fnbrno.cz 

2 



3 

Department of Clinical Haematology, University Hospital and Faculty of Medicine, 


Background: The technological development of flow cytometry (FC) together with new 

findings reveal the need for immunophenotyping in monoclonal gammopathy (MG) cases 

because of its diagnostic, prognostic and predictive significance (Paiva et al., 2010). The 


European Myeloma Network (EMN) was working on standardization of this analytical 

method and its implementation into routine clinical examination of MG cases (Rawstron 

et al., 2008). Thus a uniform protocol for the analysis of plasma cell dyscrasias (PCD) 

developed by EuroFlow consortium is widely recommended for MG analyses, especially 

in clinical studies (van Dongen et al., 2012). 

Aim: Analysis of different MG cases using Euroflow settings and validation/verification of 

this method for routine analyses. 

Subjects and methods: There were analysed 104 newly diagnosed MGs included 62 

multiple myeloma (MM) patients and 42 subjects with monoclonal gammopathy of 

undetermined significance (MGUS). Two 8-colours PCD tubes were prepared according 

to EuroFlow standard operation procedure (SOP) and analysed on flow cytometer used 

EuroFlow settings. Acquired data were reanalysed by Infinicyt software and compare 

with common analyses done in laboratory. 

Results: Using EuroFlow approach for MG analyses did not show any significant 

difference when compared to common plasma cell (PC) analyses. PCs were detectable, 

although CD138-Pacific Orange and lambda-APCH7 were not very bright in some cases. 

Merging of standardized data from two 8-colours PCD tubes increased number of 

analysed PCs what could overcome problem with low PC percentage, but a problem with 

clonality assessment remains. When other MGs than MM are analysed (MGUS with low 

PC infiltration, primary amyloidosis, lymphoplasmocytic lymphoma etc.) and different 

PC/plasmablast/B cell subpopulations should be detected, common analysis provides 

better identification of clonal cells. On the other hand, EuroFlow approach brings more 

information than common analysis and allows detection of specific PC phenotype. The 

important disadvantage of this approach is the price of some monoclonal antibodies and 

also specific software which is relatively high and thus unavailable for small laboratories. 

Conclusion: Analyses of MGs using EuroFlow settings are useful especially for 

reference laboratories participating in clinical studies as this approach allows sharing of 

standardized data for further analyses. 


This work was supported by MSM0021622434, IGA NT12425 and GACR P304/10/1395 

grants. 

References 

1. Paiva, B., Almeida, J., Pérez-Andrés, M. et al.: Utility of flow cytometry 

immunophenotyping in multiple myeloma and other clonal plasma cell-related disorders. 

- Cytometry B Clin Cytom 78(4): 239-252, 2010. 

2. Rawstron, A.C., Orfao, A., Beksac, M. et al.: Report of the European Myeloma 

Network on multiparametric flow cytometry in multiple myeloma and related disorders. 

- Haematologica 93(3):431-438, 2008. 

3. van Dongen, J.J., Lhermitte, L., Böttcher, S. et al.: EuroFlow antibody panels for 

standardized n-dimensional flow cytometric immunophenotyping of normal, reactive 

and malignant leukocytes. - Leukemia 26(9):1908-1975, 2012. 


P65. OVULATION STIMULATION PROTOCOLS UTILIZING GNRH-ANTAGONIST/HCG, 

PROMOTE CYTOTOXIC CELL POPULATIONS, PREDOMINANT IN PATIENTS WITH 

EMBRYO IMPLANTATION COMPLICATIONS 

Jan Svoboda 1 *, Zaneta Ruzickova 1 , Lucie Cuchalova 2 , Milena Kralickova 3 , Jitka Rezacova 4 , 

Milena Vrana 5 , Anna Fiserova 1 , Jan Richter 1 , Jindrich Madar 4 

1 

Laboratory of Natural Immunity, Department of Immunology and Gnotobiology, 

Institute of Microbiology, ASCR v.v.i., Prague, Czech Republic, E-mail: svoboda@biomed. 

cas.cz 

2 

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech 

Republic, Prague, Czech Republic 

3 

Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles 

University in Prague, Pilsen, Czech Republic 

4 

The Institute for the Care of Mother and Child, Prague, Czech Republic 

5 

The Institute of Hematology and Blood Transfusion, Prague, Czech Republic 

Objective: Gonadotropin-releasing hormone (GnRH) antagonist combined with 

the human chorionic gonadotropin hormone (hCG) is commonly used in assisted 

reproduction techniques (ARTs) to induce controlled ovarian hyperstimulation (COH) 

and to synchronize oocyte maturation. While hCG is known to have immunomodulatory 

properties, we aimed to assess its effect on immunological changes, with respect to 

HLA-G binding receptors and embryo implantation success. 

Design: The study involved 103 subjects, including patients undergoing COH protocols 

(n=66), divided on the basis of the pair’s fertility disorder (FD) causes (female FD, n=29; 

male FD, n=37), and age matched healthy women (n=37). The relative distribution of T 

cell (CD3+/CD4+, CD3+/CD8+) and NK cell (CD56 bright /CD16- , CD56 dim /CD16+) populations 

was evaluated together with HLA-G ligands KIR2DL4 and LILRB1 expression by flow 

cytometry in the peripheral blood of all subjects, as well as in patient follicular fluids. 

Results: Both groups of patients exhibited a significant decrease of their CD4/CD8 index, 

a down-modulation of LILRB1-positive CD8 T cells, and increased KIR2DL4-positive NK 

cell distribution, when compared to the healthy donors. These cell population levels were 

associated with failed embryotransfers in higher incidence. We attribute these changes 

to the COH protocol, since the only significant change between the patient groups was in 

the number of cytotoxic CD56 dim NK cells (elevated in the female FD group). Patients with 

male FD causes, having an above-average CD4/CD8 index (≥3.17) and below-average 

KIR2DL4+/CD56 bright NK cell levels(≤13.3%), exhibited higher embryo implantation rates. 

Conclusion: The GnRH antagonist/hCG protocol promotes CD3+/CD8+ and KIR2DL4+ NK 

cell levels, more abundant in subjects with lower implantation rates, and thus decreases 

the embryotransfer success in otherwise fertile women. 

Acknowledgements: This work was supported by a Grant Agency of the Ministry of 

Health of the Czech Republic grant [NR/9135-3]. 


P66. IMPAIRED EXPRESSION OF CD154 IN THE CD4+ T CELLS IN CVID PATIENTS IN 

RESPONSE TO DIFFERENT STIMULI 

Olga Ticha, Marcela Vlkova 

Department of Clinical Immunology and Allergology, Faculty of Medicine, Masaryk 

University and St Anne’s University Hospital, Brno, Czech Republic; olga.ticha@fnusa.cz 

Patients with common variable deficiency (CVID) is heterogeneous group of disorders 

characteristic with impairment in specific antibody production and decreased levels 

of some or all immunoglobulin isotypes in serum. Underlying genetic defects are not 

yet well defined. Apart from defects in B-cell function one of possible mechanisms 

could be failure in T cell - B cell activation and interaction. The basic for production of 

immunoglobulin is interaction between costimulatory molecule CD154 (CD40L) on the 

CD4+ T cells and molecule CD40 on the B cells. CD40L is expressed on the surface of 

helper T cells after activation by antigen and costimulators. CD40L on the activated T 

helper cell then binds to CD40 on antigen activated B cells and initiates B cell proliferation 

and differentiation and production of immunoglobulins. 

We studied expression levels of molecules CD69 and CD154 (CD40L) on surface of 

CD3+8- lymphocytes of CVID patients in response to stimulation with ionomycine (IM) 

and phorbol myristate acetate (PMA) and on surface of CD3+4+ lymphocytes in response 

to stimulation with anti-CD3 and anti-CD28 monoclonal antibodies in CVID patients 

compare to group of healthy controls. Isolated peripheral blood mononuclear cells were 

used in our experiment. 

After 4 hour stimulation with IM and PMA the percentage of CD3+CD8- lymphocytes 

which expressed costimulatory molecule CD154 on their surface was comparable in 

both groups. But statistically significant difference in percentage of activated CD3+8- 

lymphocytes expressing CD69 in both groups was found. 

On the contrary CVID patients in our cohort show statistically significant decrease in 

level of activation of CD3+CD4+ lymphocytes and decreased percentage of CD3+8+ 

expressing costimulatory molecule CD154 on their surface after stimulation with anti- 

CD3 together with anti–CD28 monoclonal antibody after 24 hours. 

We identified group of patients with low percentage of CD3+4+ lymphocytes expressing 

molecule CD154 (< 20% from CD3+4+) after anti-CD3 and anti-CD28 antibodies which 

are divided in to two different subgroups based on expression of CD154 after stimulation 

with IM and PMA - with normal or decreased levels compare to controls although all of 

them have decreased percentage of CD3+8-CD69+ lymphocytes. It is possible that this 

is caused by different underlying defect in signaling in the upper levels of activation 

pathway. 

Through the fact that defect of molecule CD154 is not described as a fundamental 

underlying cause for impaired antibody production in CVID patients our results confirm 

previously published findings showing that for some patients decreased expression 

or activation of this molecule after stimulation could be crucial. We denoted concrete 

patients whose molecules in activation signaling pathways should be studied closely for 

phosphorylation and expression levels. 



This work was supported by IGA NT/11414-5, IGA NT/1327 

References 

Costimulatory molecules and cytokine production by T lymphocytes in common variable 

immunodeficiency disease. Pons J, Ferrer JM, Martínez-Pomar N, Iglesias-Alzueta J, 

Matamoros N. Scand J Immunol. 2006 May;63(5):383-9. 

B-cell-T-cell activation and interaction in common variable immunodeficiency. Rezaei 

N, Wing JB, Aghamohammadi A, Carlring J, Lees A, Asgarian-Omran H, Pourpak Z, 

Sarrafnejad A, Kardar GA, Shahrestani T, Masoumi F, Zare A, Saghafi S, Sarrafzadeh S, 

Foster RA, Heath AW, Read RC. Hum Immunol. 2010 Apr;71(4):355-62. 

P67. ASSESSMENT OF KINETICS OF LEUKEMIC BLAST ELIMINATION DURING THE 

INDUCTION TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA USING MULTICOLOR 

FLOW CYTOMETRY 

M. Twardoch 1 , A. Sonsala 1 , J. Bulsa 1 , I. Malinowska 2 , U. Małek 3 , J. Trelińska 4 , 

E. Niedzielska 5 , K. Derwich 6 , W. Badowska 7 , M. Niedźwiecki 8 , G. Sobol 9 , 

K. Muszyńska-Rosłan 10 , M. Wysocki 11 , G. Karolczyk 12 , M. Wieczorek 13 , 

T. Urasiński 14 , J. Kowalczyk 3 , B. Mazur 15 , T. Szczepański 1 , Ł. Sędek 1 

1 

Department of Pediatric Hematology and Oncology, Medical University of Silesia, 

Zabrze, Poland; hajdix@gmail.com 

University Departments and Hospitals of Polish Pediatric Leukemia and Lymphoma 

Study Group: 

2 

Warszawa, 3 Lublin, 4 Łódź, 5 Wrocław, 6 Poznań, 7 Olsztyn, 8 Gdańsk, 9 Katowice, 

10 

Białystok, 11 Bydgoszcz, 12 Kielce, 13 Chorzów, 14 Szczecin; 

15 

Department of Microbiology and Immunology Medical University of Silesia, Zabrze, 

Poland 

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. ALL 

comprises about 80% of childhood leukemias in Poland (Kowalczyk, 2011). The level of 

minimal residual disease (MRD) is generally considered as a major prognostic factor for 

monitoring patients with ALL during the treatment (Szczepański, 2007). Evaluation of 

the MRD level is used for risk group stratification and thereby allows applying effective 

treatment protocols in these patients (Conter et al., 2010; Schrappe et al., 2011). 

The study purpose was to compare the levels of residual disease on days 15 and 33 

of treatment in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and T-lineage 

acute lymphoblastic leukemia (T-ALL). 

We studied 439 patients with ALL (384 with BCP-ALL and 55 with T-ALL), treated in 

the centers of the Polish Pediatric Leukemia/Lymphoma Study Group. Bone marrow 

samples were stained at diagnosis of leukemia with 8-color monoclonal antibody panels 

developed by the EuroFlow consortium (van Dongen et al., 2012). Precise leukemia 

associated immunophenotype was determined for each patient which facilitated their 

follow up at days 15 and 33 of induction treatment. The samples were acquired with the 


use of BD FACS Canto II flow cytometer. Subsequently, the percentage of leukemic cells 

in bone marrow was assessed using Infinicyt software. 

The mean percent of blast cells in the bone marrow of patients with BCP-ALL on day 15 

and 33 of treatment was 3.80% and 0.53%, respectively. In turn, in T-ALL patients, the 

mean percent of blast cells on day 15 and 33 of treatment reached 18.94% and 3.46%, 

respectively. 

Our study demonstrates that the levels of MRD in patients with T-ALL both at day 15 and 

33 of induction treatment are significantly higher (p

P68. SUBPOPULATIONS OF B LYMPHOCYTES - BASIC FLOW CYTOMETRIC ANALYSIS 

Vlas T., Gutová V., Panzner P. 

Department of Immunology and Allergology, Charles University in Prague, Faculty of 

Medicine and University Hospital in Pilsen, Pilsen, Czech Republic; vlast@fnplzen.cz 

B lymphocytes are an important group of white blood cells. B lymphocytes in their lives 

go through many changes. These changes are reflected by different expression of CD 

markers on the cell membrane. Presence of different CD markers divides B lymphocytes 

into several groups. Most frequent diseases with changes in subpopulations of B cells 

are common variable immunodeficiency (CVID) and selective IgA deficiency (IGAD). 

Changes in subpopulations are important in CVID for Freiburg classification. Detailed 

analysis of B lymphocyte abnormalities, and their interrelations and association with 

clinical symptoms could contribute to clarifying the causes of these diseases. 

Material and Methods: 

We analyzed 40 samples of heparinised blood. Samples for analysis were separated 

on a density gradient (Histopaque 1077, SigmaAldrich, UK). Using flow cytometry we 

determined the percentage of cells expressing different CD molecules (CD19, CD21, 

CD27, CD38, IgM and IgD). Monoclonal antibodies used Anti-HU CD19 PE-DyLight 594, 

Anti-HU CD21 FITC, Anti-HU CD27 PE-Cy5, Anti-HU CD38 PE-Cy5, Anti-HU IgM PE (all from 

Exbio, CZE) and Anti-HU IgD FITC (Beckmann Coulter, USA).The analysis was performed on 

flow cytometer FC500 (Beckmann Clouter, USA) with software CXP analysis (Beckmann 

Coulter, USA). 

Results: 

We found a significant increase by 26.4% (p

Conclusion: 

Analysis by flow cytometry is a quick indication of subpopulations of B lymphocytes. 

Analysis by flow cytometry allows the inclusion of the patient in the group of Freiburg 

classification. The method is ready for clinical use. 

Literature: 

Annick A.J.M. van de Ven, Lymphocyte characteristics in children with common variable 

immunodeficiency, Clinical Immunology, Volume 135, Issue 1, April 2010, Pages 63-71, 

ISSN 1521-6616 

Daniele Moratto, Anna Virginia Gulino, Combined decrease of defined B and T cell 

subsets in a group of common variable immunodeficiency patients, Clinical Immunology, 

Volume 121, Issue 2, November 2006, Pages 203-214, ISSN 1521-6616 

Jimmy Ko, Lin Radigan, Immune competence and switched memory B cells in common 

variable immunodeficiency, Clinical Immunology, Volume 116, Issue 1, July 2005, Pages 

37-41, ISSN 1521-6616 

Jens Thiel, Lucas Kimmig, Genetic CD21 deficiency is associated with 

hypogammaglobulinemia, Journal of Allergy and Clinical Immunology, Volume 129, Issue 

3, March 2012, Pages 801-810.e6, ISSN 0091-6749 

J. Smet, F. Mascart, Are the reference values of B cell subpopulations used in adults for 

classification of common variable immunodeficiencies appropriate for children?, Clinical 

Immunology, Volume 138, Issue 3, March 2011, Pages 266-273, ISSN 1521-6616 

P69. FLOW CYTOMETRIC ASSESSMENT OF COAGULATION SYSTEM ACTIVATION BY 

TISSUE FACTOR IN PREGNANCY COMPLICATIONS 

Martin Novák 2 , Martin Procházka 1 , Jana Procházková 2 , Marek Ľubušký 1 , Petr Polák 3 , 

Luděk Slavík 2 , Jana Úlehlová 2 , Vít Procházka 2 , Radovan Pilka 1 

1 

Department of Obstetrics and Gynecology, Palacký University Faculty of Medicine and 

Dentistry and University Hospital Olomouc, Czech Republic 

2 

Department of Hemato-Oncology, Palacký University Faculty of Medicine and Dentistry 

and University Hospital Olomouc, Czech Republic, novak.m@email.cz 

3 

Department of Obstetrics and Gynecology, Palacký University Faculty of Medicine 

and Dentistry and University Hospital Olomouc, and U.S.G.POL Antenatal Clinic, Czech 

Republic 

One part of a project to identify the causes of preeclampsia and other obstetric 

complications was aimed at determining the impact of tissue factor on activation of 

these pathophysiological processes. Based on the latest pathophysiological findings, the 

objective was to find markers relevant for determining the role of individual processes in 

clinical expression of these conditions. 

Tissue factor (TF, CD142) is an integral membrane protein constitutively expressed in 

many types of cells of the vascular system but not normally expressed on cells in contact 

with flowing blood 1 . It is a specific receptor with a high affinity for factor VII / VIIa and 

acts as cofactor for factor VIIa. Exposure of activated TF in the coagulation system 


triggers a process of normal blood clotting and thrombosis in numerous thrombotic 

disorders. Blood components and especially monocytes are unable to constitutively 

express functional TF; however, they are capable of synthesizing and expressing TF when 

stimulated, either by lipopolysaccharides or inflammatory cytokines 2 . Expression of TF 

on monocytes participates in the mechanism of the development of thrombosis in many 

conditions, including sepsis, atherosclerosis, use of oral contraceptives, cancers and 

hyperhomocysteinemia. 

According to the latest findings, activation of hypercoagulation is significantly contributed 

to by production of autoantibodies. As soon as this mechanism is activated, the antibody 

induces expression of TF on monocytes or vascular endothelial cells. However, there is 

increasing evidence that certain types of antiphospholipid antibodies, in particular those 

against beta-2-glykoprotein I (beta-2GPI), stimulate expression of TF on monocytes 3,4 . 

Therefore, we proposed a model of cytometric analysis of coagulation system 

activation in preeclampsia and other complications in pregnancy using expression of 

TF on monocytes and assessment of TF-induced generation of thrombin in plasma. To 

determine expression of tissue factor (CD142) on monocytes, multicolor flow cytometry 

was used with the antibodies anti-CD45 PerCP, clone MEM-28 (Exbio Prague), anti-CD14 

APC, clone MEM-15 (Exbio Prague), CD16b FITC, clone MEM-154 (Exbio Prague) and anti- 

CD142 PE, clone HTF-1 (BD Pharmingen), and relevant isotope controls. The analyses 

were carried out using samples of peripheral blood stabilized with K3EDTA. Material 

that could not have been processed within two hours from sampling was fixed with 

the TransFix reagent (Cytomark, Caltag- Medsystems Ltd). Samples were incubated with 

antibodies according to the manufacturer’s recommendation, processed using a lyse/ 

no-wash procedure and analyzed with the BD Biosciences FACSCalibur flow cytometer 

and CellQuest Pro software. For the analysis, a minimum of 5,000 events were acquired 

within the CD45+/CD14+ gate. 

The model was verified in patients with severe antiphospholipid syndrome and a high 

expression of antibodies, especially against beta-2GPI. As a result, expression of TF on 

monocytes was completely inhibited and thrombin generation in plasma was markedly 

decreased. In the light of these findings, the detection of CD142 expression using flow 

cytometry methods appears to be an effective approach to monitoring of coagulation 

system activation and the reported method seems to be very promising for further study 

of mechanisms underlying preeclampsia and the related conditions. 


This work was supported by the grant IGA NT 14394 and student project LF-2013-004 of 

Palacký University. 

References 

1. Roubey RAS. Tissue factor pathway and the antiphospholipid syndrome. J Autoimmun. 

2000;15: 217-220. 

2. Gregory SA, Morrissey JH, Edgington TS. Regulation of tissue factor gene expression 

in the monocyte procoagulant response to endotoxin. Mol Cell Biol. 1989;9:2752-2755 

3. Amengual O, Atsumi T, Khamashta MA, Hughes GRV. The role of the tissue factor 

pathway in the hypercoagulable state in patients with the antiphospholipid syndrome. 

Thromb Haemost. 1998;79:276-281. 

4. Reverter JC, Tassies D, Font J, et al. Effects of human monoclonal anticardiolipin 


antibodies on platelet function and on tissue factor expression on monocytes. Arthritis 

Rheum. 1998;41:1420-1427. 

P70. FLOW CYTOMETRIC ANALYSIS OF WHITE BLOOD CELLS IN HUMAN PERIPHERAL 

BLOOD 

J. Mlynárová 1 , P. Musil 1 , D. Kučerová 1 , J. Kyselovič 1,2 

1 

Faculty of Pharmacy, 

2 

Faculty of Medicine, Comenius University, Bratislava, Slovak Republic 

Email: jnmlynarova@gmail.com 

The purpose of our work was to collect baseline data regarding feasibility and suitability 

of white blood cells isolated from human peripheral blood for further experiments (cell 

therapy). Furthermore, to analyses viability of isolated cells and identifies the number of 

total cells, leukocytes, T cells and monocytes. 

Peripheral blood was collected from four volunteers at National transfusion service 

in Bratislava, Slovakia. After proper blood processing, resting buffycoat was used for 

white blood cells isolation by gradient centrifugation with Histopaque solution. Layer 

containing WBC was collected and analyzed by flow cytometry. CD45, CD3, CD4 markers 

were used to identify leukocytes and T cells. Antibody cocktail containing C14, CD15 and 

CD45 was used to identify monocytes. 

We isolated 5,6x10 7 ± 1,4x10 7 total cells with 99±0,2% viability. 5x10 7 ±1,4x10 7 cells 

(89±14% of total cell number) were viable CD45 + . From CD45 + cells we identified in 

total 2,6x10 7 ±9,3x10 6 CD3 + cells. From the total number of CD3 + cells, we identified 

1,4x10 7 ±3,2x10 6 viable CD3 + /CD4 + cells. The total number of monocytes in our samples 

was in average 1,3x10 7 ±6,4x10 6 . 

In conclusion, we found a very high viability of cells after isolation process and identified 

several leukocyte populations in buffycoat samples acquired from peripheral blood. 


This work was supported by grant ITMS 26240220071. Buffycoat was collected at 

National transfusion service in Bratislava, Slovakia. 

P71. FLOW CYTOMETRY ANALYSIS OF PLASMA CELL PHENOTYPE IN 

EXTRAMEDULLARY RELAPSE OF MULTIPLE MYELOMA 

Lucie Rihova 1,2 , Renata Suska 1 , Hana Svachova 2 , Pavla Vsianska 1,2 , Zdenka Pavkova 1,2 , 

Renata Bezdekova 2 , Petra Kucerova 1 , Sabina Sevcikova 2 , Ludek Pour 2,3 , Roman Hajek 1,2,4 

1 

Department of Clinical Haematology, University Hospital Brno, Brno, Czech Republic; 

lucie.rihova@fnbrno.cz 

2 




3 

Department of Internal Medicine - Haematology, University Hospital Brno and Faculty 

of Medicine, Brno, Czech Republic 

4 

Department of Clinical Haematology, University Hospital and Faculty of Medicine, 


Background: Multiple myeloma (MM) is the second most common haematological 

malignancy in the world which is characteristic by a presence of clonal plasma 

cells (PCs). The introduction of new drugs (thalidomide, bortezomib, revlimid) has 

dramatically improved survival of MM patients, but MM still remains an incurable 

disease. Unfortunately, an increase in the incidence of extramedullary relapse of MM 

(EM), an aggressive mostly resistant entity with abysmal prognosis for patients has 

been reported. EM can affect any area of tissue - soft tissue involvement can be with or 

without relationship to bone marrow (BM). Finding a marker and/or phenotype allowing 

predict a risk of EM formation could help to improve treatment strategies and prolong 

patient life. 

Aim: Analysis and comparison of PC phenotype in bone marrow and tumor tissue (TU) 

of EM cases. 

Subjects and methods: There were analysed 27 MM patients (median age 62 years) 

with confirmed EM. Analysis of 24 bone marrows and 18 tumor tissues was done by 

polychromatic flow cytometry. Phenotype of CD38 + CD138 + PC was analysed using CD19, 

CD20, CD27, CD28, CD44, CD56, CD81, CD117 and nestin. PCs from BM and/or TU were 

considered positive for given marker when its expression on PCs was higher than 20%. 

Results: There were detected CD38 + CD138 + PCs in all BM samples and in almost all TU 

samples (except 1 sample probably acquired not from tumor site). PC infiltration was 

significantly higher in TU then in BM [median of infiltration 51.7% (range 0.0-94.3) vs. 

1.5% (0.01-93.9); p

P72. IMPACT OF HISTONE DEACETYLASE INHIBITORS ON ANTICANCER EFFECTS OF 

CYTOSTATICS 

Tomáš Groh 1 , Helena Maříková 2 , Jan Hraběta 2 , Ashraf M. Khalil 2 , Tomáš Eckschlager 2 , 

Marie Stiborová 1 

1 

Department of Biochemistry, Faculty of Science, Charles University, Prague 

2 

Department of Pediatric Hematology and Oncology, 2 nd Medical School, Charles 

University, Prague 

grohtomasmail@gmail.com 

During the last few decades, several approaches have been applied in effort to discover 

more effective anticancer drugs. Many promising compounds have been investigated. 

However, chemoresistance is one of the main causes of failure of treatment. Epigenetic 

changes are the changes of gene expression or cellular phenotype caused by mechanisms 

other than are the changes in DNA sequence. Histone acetylation has been investigated 

as therapeutic target because of its importance in regulation of gene expression. 

Different histone deacetylase (HDAC) inhibitors induce death of cancer cells by different 

mechanisms that include changes in gene expressions and alterations of both histone 

and non-histone proteins. Enhanced histone acetylation in tumors results in modification 

of expression of genes involved in cell signaling. Inhibition of HDACs causes changed 

expression of 2-10 % of genes involved in important biological processes. Inhibition 

of HDACs leads to changes in chromatin architecture that can result in modulation of 

gene transcription. Inhibitors of HDACs have also an impact on non-histone proteins. 

They can change their stability and interaction with other proteins or with DNA. The 

results of experiments and clinical studies have demonstrated that combination of 

HDAC inhibitors with some anticancer drugs have synergistic or additive effects. Valproic 

acid (VPA) is known as an anticonvulsant used for a treatment of bipolar disorder and 

epilepsy. Last decades, VPA is also studied as an inhibitor of histone deacetylases. Beside 

VPA, trichostatin A (TSA) is another compound utilized as an HDAC inhibitor. 

We tested the effect of non-toxic concentrations of VPA and TSA on cytotoxicity of 

different cytostatics to human neuroblastoma cells. VPA increases cytotoxicity of 

etoposide, cisplatin but not vincristine in co-cultivation experiments. Similar, but lower 

effects exhibited TSA. Valpromide, an analogue of VPA that lack the histone deacetylase 

inhibition effect, did not increase cytotoxicity of etoposide and cisplatin. Inhibition of 

histone deacetylases by VPA seems to influence cell proapoptotic signals or regulate 

reparation machineries of neuroblastoma cells. We have found that VPA increased 

cytotoxicity of etoposide to neuroblastoma cells in vitro, if etoposide was co-cultivated 

with VPA, but preincubation of these cells with VPA did not increase the etoposide 

efficacy. We observed the same trend using cisplatin. The results provide additional 

evidence for the use of VPA in complex anticancer therapy. 


This work was supported by GAUK635712, UNCE204025/2012 and P301/10/0356. 


P73. THE V-ATPASE-INHIBITOR BAFILOMYCIN A ABROGATES ELLIPTICINE 

CHEMORESISITANCE IN NEUROBLASTOMA CELLS 

Hraběta J. 1 , Maříková H. 1 , Uhlík J. 3 , Stiborová M. 2 , Groh T. 2 , Eckschlager T. 1 

1 

Department of Pediatric Hematology and Oncology, 2 nd Medical School, Charles 

University and University Hospital Motol, Prague, Czech Republic; 

janhrabeta@gmail.com 

2 

Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech 

Republic; 

3 

Department of Histology and Embryology, 2 nd Medical School, Charles University 

Prague, Czech Republic 

Ellipticine, an alkaloid isolated from Apocyanaceae plants, and several of its more 

soluble derivatives exhibit significant antitumor activities. The main reasons for the 

interest in ellipticine and its derivatives for clinical purposes are their high efficiencies 

against several types of cancer, their rather limited toxic side effects, and their 

complete lack of hematological toxicity. Previously, we determined that ellipticine 

was able to induce resistance in the neuroblastoma cell line UKF-NB-4. Resistance 

was caused by a combination of overexpression of Bcl-2, efflux or degradation of the 

drug, and downregulation of topoisomerases. Upregulation of enzymes of oxidative 

phosphorylation, cellular respiration and V-ATPases (Procházka et al., 2012). Vacuolar 

(V)-ATPase is the enzyme complex necessary for the acidification of some intracellular 

compartments (trans-Golgi network, endosomes, lysosomes, secretory granules) and 

protonated chemicals may be trapped into these vacuoles at low pH, due to the pKa value 

of the chemical and difference of pH across the cell membrane (Spugnini et al.,2010).The 

aim of this study was to investigate the importance of V-ATPases in resistance UKF-NB4 

cells to ellipticine. 

Ellipticine treatment is a causes of significant and concentration-dependent cytoplasmic 

vacuolization of the neurobalstoma cells. The vacuoles were detectable already 30 

minutes after the treatment and ellipticine was concentrated to the vesicular structures 

in cytoplasm. Vacuolization and intravesicular ellipticine-associated fluorescence was 

abolished by co-treatment with the specific V-ATPase inhibitor bafilomycin A1. Ellipticine 

exposed cells were analysed by immunoblotting for autophagy marker MAP1 LC3. 

Increased expresion of LC3 II persisted even after 24h. However, in transmission electron 

microscopy, we observed dilatation and multiplication of lysosomes without the of share 

autophagosomes. Bafilomycin A1 pretreatment increased the efficiency of ellipticine to 

neuroblastoma cells and abolish of resistance. Bafilomycine pretreatment markedly 

increased formation of covalent ellipticine-derived DNA adducts. 

Ellipticine is sequestrated to the vesicular structures of lysosomal origin. Specific 

Vacuolar-ATPase inhibition increased of formation ellipticine-DNA adducts, markedly 

increasaed of ellipticine anticancer action and abolish chemoresistance. 


Supported by the Grant Agency of the Czech Republic (grant P301/10/0356) 

References 


Procházka P, Libra A, Zemanová Z, Hřebačková J, Poljaková J, Hraběta J, Bunček 

M, Stiborová M, Eckschlager T. Mechanisms of ellipticine-mediated resistance in 

UKF-NB-4 neuroblastoma cells. Cancer Sci. 2012;103:334-41. 

Spugnini EP, Citro G, Fais S. Proton pump inhibitors as anti vacuolar-ATPases drugs: a 

novel anticancer strategy. J Exp Clin Cancer Res. 2010;29:44. 

P74. NESTIN EXPRESSION IN LEUKEMIA CELLS. 

Tomáš Loja 1,2 , Marek Borský 1 , Tomáš Bernard 1 , Michael Doubek 1,2 

1 

Department of Internal Medicine - Hematology and Oncology, University Hospital Brno 

and Faculty of Medicine, Masaryk University, Czech Republic; tomas.loja@gmail.com 

2 

CEITEC - Central European Institute of Technology, Masaryk University, Czech Republic 

Protein nestin is considered a marker of immature (stem/progenitor) cells and its 

expression occurs during mammalian embryogenesis. Re-expression of nestin in adults 

occurs in various pathologic conditions; neoplastic transformatio is one of them. Nestin 

has been detected in whole range of solid tumors where its expression correlates with 

tumor malignancy and invasiveness - nestin expression levels are higher in tumors with 

high grades than in those with low grades. Therefore, nestin is thought to be negative 

prognostic marker. In addition, some nestin-positive populations of tumor cells have the 

same features as cancer stem cells (CSCs) responsible for tumor relapse according to 

CSCs hypothesis. So far it is very little known about nestin expression in leukemia cells. 

Our pilot data proved nestin in tumor cells of various leukemia and lymphoma cell lines, as 

well as in patients with chronic lymphocytic leukemia (CLL) and other hematooncological 

diagnoses. CLL, the most common adult leukemia in Western countries, is a progressive 

hematopoietic disorder characterized by clonal proliferation and accumulation of 

neoplastic mature-appearing B-lymphocytes in the blood, bone marrow, spleen, and 

lymph nodes. Malignant lymphoproliferative diseases represent a group of cancers 

with high incidence, variable biological behavior and prognosis, therefore, attention is 

paid to new prognostic factors, which would allow to stratify patients according to their 

individual risk factors and to optimize patients treatment. 

Nestin expression level is linked to tumor subtypes which are less differentiated, more 

aggressive, and have poorer prognosis; therefore, determination of nestin expression in 

CLL patients as well as in other hematological malignancies could be used to estimate 

the biological behavior. In addition, combined analysis of nestin and other diagnostic/ 

prognostic factors may help to improve stratification and therapy of oncologic patients. 


This work was supported by the project (Ministry of Health, Czech Republic) for 

conceptual development of research organization 65269705 (University Hospital 

Brno, Brno, Czech Republic). 


P75. 5-FLUOROURACIL-SELECTED TUMOUR CELLS EXERT CROSS-RESISTANCE WHICH 

CAN BE MODULATED BY PHARMACOLOGICAL AND MOLECULAR INHIBITION OF 

ALDEHYDE DEHYDROGENASE 

Miroslava Matuskova, Erika Durinikova, Lucia Kucerova, Zuzana Kozovska 

Cancer Research Institute of Slovak Academy of Sciences, Bratislava Slovak Republic 

One of the obstacles of effective cancer treatment is selection for chemo-resistant subpopulations 

of malignant cell during chemotherapy. Innovative approaches need to be 

developed to affect malignant cells resistant to conventional treatment. Chemo-resistant 

tumour cells use more mechanisms to escape cytotoxic effect of drugs. Increased 

expression of aldehyde dehydrogenase (ALDH), particularly 1A1 isoform (ALDH1A1) is 

also involved in drug resistance (Januchowski et al., 2013). 

We aimed to evaluate the sensitivity of 5-Fluorouracil (5-FU) resistant tumour cells to 

panel of drugs, and prove the effect of inhibition of ALDH to evaluate its role in response 

to chemotherapy. 

Model chemo-resistant tumour cell line HT-29/EGFP/FUR proliferating in clinically 

relevant plasma concentration of 5-FU was derived from human colon carcinoma cells 

HT-29 by long-term cultivation in sequentially increasing 5-FU concentration. Tumour 

cells were retrovirally transduced with enhanced green fluorescent protein (EGFP) for 

unequivocal identification in cytometric experiments and evaluation of sensitivity to 

chemotherapy treatment by fluorimetric assay. Immunophenotype, ALDH activity and 

apoptosis were determined by flow cytometry. Kinetic experiments were performed 

using Incucyte device. Gene expression was evaluated by qPCR, siRNA was delivered 

by nucleofection. Athymic mice were used for experiments in vivo. 

We demonstrated altered expression of enzymes involved in nucleotide metabolism 

and transporter protein ABCC4. Changes correlated with their suggested role in 5-FU 

resistance. Differences in expression of stem cells` markers ALDH1A1 and CD133 were 

observed. We expected increased expression of ALDH1A1 enzyme in chemo-resistant 

derivate of HT-29 cells. The functional assay ALDEFLUOR confirmed increased activity 

of ALDH however ALDH1A1 – specific qPCR showed decreased expression of 1A1 isoform. 

Lower expression of ALDH1 was also confirmed on protein level by Western-blotting. 

Other ALDH isoforms have probably increased expression. HT-29/EGFP/FUR cells were 

cross-resistant to Cis-Platin, Paclitaxel, Doxorubicine and Cyclofosamide, .which are 

structurally and functionally unrelated to 5-FU. HT-29/EGFP/FUR derived xenografts 

maintained their chemo-resistance for several months without selection pressure 

of 5-FU, but exerted lower tumorigenicity. MSC co-injection had tumour-favouring 

potential on the HT-29/EGFP/FUR xenograft growth. Immunomagnetically separated 

CD133-positive subpopulation showed decreased sensitivity to chemotherapy. 

ALDH activity was pharmacologically inhibited by diethylaminobenzaldehyde (DEAB) 

or by RNA interference using set of ALDH1A1 specific small interfering RNA (siRNA). 

Nucloefection of ALDH1A1 siRNA has short term effect on target genes’ expression. 

Decreased level of mRNA was observed up to 72 hours post transfection. Tumour cells with 

inhibited ALDH activity exerted altered sensitivity to 5-FU, Cis-platin and oxidative stress. 



This work was supported by Slovak Cancer Research Foundation, VEGA grants 2/0171/13, 

2/0130/13, 2/0088/11 and by Slovak Research and Development Agency under the 

contract No. APVV-0230-11. 

References 

Januchowski, R, Wojtowicz, K, and Zabel, M (2013): The role of aldehyde dehydrogenase 

(ALDH) in cancer drug resistance. Biomed Pharmacother. [Epub ahead of print] 

P76. CYTOKINE DYNAMICS AND IMMUNE CELLS PROPORTION DURING TUMOUR 

GROWTH AND SUBSEQUENT EXCISION IN LEWIS RAT SARCOMA MODEL 

Mishra R. 1,2 , Horák V. 1 , Kovalská J. 1,2 and Rajmon R. 2 

1 

Institute of Animal Physiology and Genetics, AV CR v.v.i., 277 21 Liběchov, Czech 

Republic; mishra@iapg.cas.cz 

2 

Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural 

Resources, 160 00 Prague, Czech Republic 

The slow rate of systemic treatment of sarcoma is still a big challenge for researchers. 

Among other methods, immunotherapy is one of the effective methods for the treatment 

of patient with metastatic sarcoma (R. G.Maki; 2001, 2006). Rat sarcoma model has been 

established in our laboratory in which tumour progression and regression occur and are 

associated with distinct immunological changes (Holubova et al; 2012). 

Moreover, changes in proportion of immune cell subpopulations in peripheral blood can 

bring important information about tumour growth, its immunogenicity and efficiency 

of therapy. In this study, our aim was to study changes in immune cell populations 

and cytokine profile during tumour growth, after tumour excision and after second 

inoculation by initially used cell line. 

Thirty one Lewis rats (15 males and 16 females) were inoculated subcutaneously with the 

R5-28-2 rat sarcoma cells (D6 clone). Four males and four females without cell inoculation 

were used as controls. Tumours appeared in all rats 2 weeks after cell inoculation. They 

were excised at the 5 th week reaching the average size around 35 mm. At the 11 th week 

of experiment, second inoculation of sarcoma cells was done and rats were monitored 

for next 4 weeks. Peripheral blood was taken once a week to determine haematological 

parameters, proportion of lymphocyte subpopulation and to isolate serum for cytokine 

screening and quantification. 

Number of leukocytes increased with growing tumour size due to expanded population 

of granulocytes. Four kind of immune cells (CD4 + , CD8 + , CD161 + and RT1B + ) were 

analysed by flow cytometry. Proportion of CD4 + and CD8 + cells decreased with tumour 

growth. Interestingly, these changes were more prominent in females than in males. 

Increased serum levels of TNF-α, ICAM-1, MCP-1, IL-6 and L-Selectin were also observed 

in tumour-bearing animals. Excision of tumour normalized all the parameters. The 

second inoculation of sarcoma cells did not result tumour formation and changes in any 

of the studied parameters. 


We conclude that decrease in the population of CD4 + and CD8 + cells along with alterations 

in cytokine profiling could play an important role in tumour growth. Moreover, failure of 

tumour development after second inoculation provides a clue towards induction of antitumour 

immune memory caused by the first inoculation. In this study we characterized 

leukocyte populations and cytokine expression in the rat sarcoma model that will be 

used for study of mechanism of anti-tumor immunity as well as development of new 

tumor therapy. 

Acknowledgment 

This work was supported by MEYS CR (CZ.1.05/2.1.00/03.0124) and by RVO (67985904). 

References 

Holubova, M., Leba, M., Sedmikova, M., Vannucci, L., Horak, V.: Characterization of three 

newly established rat sarcoma cell clones. In Vitro Cell Dev Biol, 48(10):610-8, 2012. 

Maki, R.G.: Soft tissue sarcoma as a model disease to examine cancer immunotherapy. 

Current Opinion in Oncology, vol. 13, no. 4, pp. 270–274, 2001. 

Maki, R.G.: Future directions for immunotherapeutic intervention against sarcomas. 

Current Opinion in Oncology, vol. 18, no. 4, pp. 363–368, 2006. 


Figure 1 showing dot plot for CD4 + cells and Granulocytes. Figure 1a, 1b and 1c is dot 

plot for week 0, week 5 th and week 15 th respectively. From figure it is evident that with 

growing tumor size, proportion of CD4 + cells decreasing and of granulocytes increasing, 

and after excision at 5 th week it came almost to normal level and didn’t increase further 

after four week of second inoculation. 


P77. DNA DAMAGE RESPONSE IN HEMATOPOETIC CELLS OF ACUTE LYMPHOBLASTIC 

LEUKEMIA PATIENTS 

Lenka Vokalova 1 , Michaela Fajtova 1 , Katarina Vyparinova 1 , Alexandra Somsedikova 1 , 

Pavol Kosik 1 , Judita Puskacova 2 , Alexandra Kolenova 2 , Eva Markova 1 , Igor Belyaev 1 

1 

Cancer Research Institute, Bratislava, Slovak Republic; igor.beliaev@savba.sk 

2 

Children´s Hematology and Oncology Clinic, Faculty of Medicine, The Comenius 

University, Bratislava, Slovak Republic 

Leukemia is a clonal disease arising from the neoplastic transformation of a single 

hematopoietic CD34 stem/progenitor cell which via repeated cell division generates 

an expanding clone of neoplastic cells, which is highly malignant in acute leukemias. 

DNA damage and apoptosis are key elements in the accumulation of changes associated 

with cell transformation. Leukemic cells may significantly differ in their sensitivity to 

therapeutical treatment. While cells from majority of B-chronic lymphoid leukemia 

patients were hypersensitive to irradiation as compared with normal lymphocytes, some 

cases were highly resistant even at high radiation doses (Blaise, Alapetite et al. 2002). The 

mechanisms of individual variations in sensitivity of leukemia patients to therapeutical 

treatment are not well defined. Specific leukemic gene fusions (LGF) may affect this 

sensitivity. For example, BCR/ABL fusion tyrosine kinase transforms hematopoietic 

stem cells causing chronic myelogenous leukemia (CML) and acute lymphoblastic 

leukemia (ALL) (Skorski 2008). However, BCR/ABL also enhances DNA damage caused by 

endogenous reactive oxygen species and modulates DNA damage response. Systematic 

analysis of DNA damage signaling in the presence of BCR/ABL thus offers opportunities 

to identify mechanisms of leukemic progression (Griaud, Williamson et al. 2012). How 

other commonly observed LGF may affect DNA damage response is not known. In 

addition, there is significant variation in immunophenotype between ALL patients which 

may also affect DNA damage response. We aim to analyze DNA damage response and 

apoptosis in cells from ALL patients with different LGF and immunophenotype. We study 

double-strand breaks (DSB) repair and apoptosis after γ-irradiation in lymphocytes or 

bone marrow from healthy subjects and ALL patients with and without LGF and different 

immunophenotype. DSB were assessed by enumeration of γH2AX and 53BP1 foci. 

Apoptosis was analyzed by quantifying the annexin V-FITC and propidium iodide signals 

using automated fluorescent microscopy (Metafer MetaCyte) and flow cytometry (BD 

FACSCanto II), respectively. Immunophenotyping was performed by flow cytometry 

using antibodies CD34, CD10, CD20, CD19, CD22, CD33. We identified time kinetics (0 

min, 30 min, 1 hour, 2hours, 18 hours) of γ H2AX/53BP1 foci after irradiation with 2 

Gy in 9 leukemic patients and compared outcomes of these kinetics with apoptosis. 

Preliminary data indicate increased level of endogenous 53BP1 and γ-radiation induced 

γH2AX and co-localized γH2AX/53BP1 foci in patients with CD34 +low as compared to CD34 

+high 

immunophenotype. Low number of studied patients with specific gene fusions (E2A- 

PBX, BCR-ABL p190, sil/tal1, TEL AML1) does not allow concluding on correlation of DNA 

damage response and presence of gene fusions. The study group should be increased to 

validate significance of the results. 



This work was supported by the Slovak Research and Development Agency (APVV 

0669-10) and the VEGA Grant Agency (2/0150/11) of the Slovak Republic. 

References 

Blaise, R., C. Alapetite, P. Masdehors, H. Merle-Beral, C. Roulin, J. Delic, et al. (2002). 

„High levels of chromosome aberrations correlate with impaired in vitro radiationinduced 

apoptosis and DNA repair in human B-chronic lymphocytic leukaemia cells.“ Int 

J Radiat Biol 78(8): 671-679. 

Griaud, F., A. J. K. Williamson, S. Taylor, D. N. Potier, E. Spooncer, A. Pierce, et al. (2012). 

„BCR/ABL modulates protein phosphorylation associated with the etoposide-induced 

DNA damage response.“ Journal of Proteomics 77: 14-26. 

Skorski, T. (2008). „BCR/ABL, DNA damage and DNA repair: Implications for new 

treatment concepts.“ Leukemia & Lymphoma 49(4): 610-614. 


Fig. 1. Immunophenotype of patient with acute B-lymphoblastic leukemia, CD34 +high 

immunophenotype. Parameter CD45 shows gated neutrophils (Neu), monocytes (Mo), 

lymphocytes (Ly) and pathologic cells (Pat). 

P78. THE DISTRIBUTION OF PROFLAVINE DERIVATIVES IN LEUKEMIA CELLS 

Zuzana Ipothova 1 , Luba Hunakova 2 , Helena Paulikova 3 , Lydia Cizekova 3 

1 

Department of Chemical and Biochemical Engineering, Faculty of Chemical and Food 

Technology, Slovak University of Technology in Bratislava, Slovak Republic; 

zuzana.ipothova@gmail.com 

2 

Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic 

3 

Department of Biochemistry and Microbiology, Faculty of Chemical and Food 

Technology, Slovak University of Technology in Bratislava, Slovak Republic 

In last two decades, new acridine derivatives have been intensively studied as novel 

antitumor drug candidates which are able to intercalate into DNA and interact with 

nuclear topoisomerase and telomerase enzymes (Cholewinski et al., 2011). Besides 

accumulation in the cell nucleus, the acridine derivatives showed also an atypical 

subcellular localization in mitochondria and lysosomes (Peixoto et al., 2009). Recently, 

cytotoxicity of novel acridine derivatives, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden) 

imino)acridine hydrochlorides (AcrDIMs), has been confirmed for leukemic cell lines 

(Janovec et al., 2011). These derivatives with different side alkyl chain (C2 –C6) showed 


ability to intercalate DNA although they possessed various affinity to DNA (in vitro 

studies). Furthermore, hexyl-AcrDIM, the derivative with weak DNA-binding affinity, had 

the most potent cytotoxic effect against several cancer cell lines. 

In our present study, we have analyzed the accumulation and distribution of AcrDIMs in 

mouse leukemic cell line L1210 to explain different cytotoxicity of analogs in the series. 

We have monitored intracellular distribution of propyl- and hexyl-AcrDIM using an Amnis 

ImageStream Imaging Flow Cytometer. Surprisingly, the colocalizations of AcrDIMs with 

nucleic acids stain SytoRed 62 have shown that studied derivatives were not accumulated 

in the nucleus even after long-term incubation (24 h). They were not localized in the 

lysosomes as well (colocalization study of the derivatives with Monodansylcadaverine). 

The fluorescence of propyl-AcrDIM was not overlaid with mitochondrial dye MitoRed, 

but the derivative with the highest lipophilicity - hexyl-AcrDIM was partially accumulated 

in the mitochondria (24 h). 

Our results have confirmed that hexyl-AcrDIM, the most cytotoxic derivative of series, 

was partially accumulated in the mitochondria. Mitochondrion, the energetic center of 

cell and a key organelle in an intrinsic apoptotic pathway, is target of many anticancer 

agents (Costantini et al., 2000). They may cause mitochondrial dysfunction and induce 

oxidative stress in the cells. The level of superoxide radical in the cells treated with 

hexyl-AcrDIM has increased several times, and block in synthetic phase of cell cycle 

and apoptotic DNA-fragmentation has occurred. Nevertheless, cytotoxic mechanism of 

hexyl-AcrDIM is not clear, yet. But the lipophilicity plays a crucial role in the distribution 

of AcrDIMs and their following anticancer effect. 


This work was supported by Structural Funds EU (ITMS: 26240220071) and the Grant 

Agency VEGA of the Ministry of Education of the Slovak Republic (1/0672/11, 2/0177/11) 

and it is result of the project implementation: TRANSMED 2, ITMS: 26240120030, 

supported by the Research & Development Operational Programme funded by the ERDF. 

References 

Costantini, P., Jacotot, E., Decaudin, D., Kroemer, G.: Mitochondrion as a novel target 

of anticancer chemotherapy. - Journal of the National Cancer Institute 92: 1042-1053, 

2000. 

Cholewinski, G., Dzierzbicka, K., Kolodziejcyk, A.M.: Natural and synthetic acridines/ 

acridones as antitumor agents: their biological activities and methods of synthesis. - 

Pharmacological Reports 63: 305-336, 2011. 

Janovec, L., Kožurková, M., Sabolová, D., Ungvarský, J., Paulíková, H., Plšíková, J., Vantová, 

Z., Imrich, J.: Cytotoxic 3,6-bis((imidazolidinone)imino)acridines: synthesis, DNA binding 

and molecular modeling. - Bioorganic & Medicinal Chemistry 19: 1790-1801, 2011. 

Peixoto, P., Zeghida, W., Carrez, D., Wu, T.D., Wattez, N., Croisy, A. Demeunynck, M., 

Guerquin-Kern, J.L., Lansiaux, A.: Unusual cellular uptake of cytotoxic 4-hydroxymethyl- 

3-aminoacridine. - European Journal of Medicinal Chemistry 44: 4758-4763, 2009. 


P79. NEW TYPE OF PHOTOSENSITIZER IS EFFECTIVE ALSO FOR THE RESISTANT 

TUMORS 

Alžbeta Grolmusová 1 , Pavel Abaffy 1 , Helena Paulíková 1 , Lýdia Čižeková 1 , Zuzana Ipóthová 2 , 

Ľubica Hunáková 3 

1 

Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, 

Slovak University of Technology, Bratislava, Slovak Republic; 

alzbeta.grolmusova@gmail.com 

2 

Department of Chemical and Biochemical Engineering, Faculty of Chemical and Food 

Technology, Slovak University of Technology, Bratislava, Slovak Republic 

3 

Cancer Research Institute, Slovak Academy of Science, Bratislava, Slovak Republic 

Photodynamic therapy (PDT) represents a new alternative for the treatment of cancer. 

PDT uses a drug, called a photosensitizer (PS), oxygen and light with appropriate 

wavelength. When photosensitizers are exposed to the light with specific wavelength, 

they produce free oxygen radicals that kill nearby cells (Robertson et al., 2009). Except 

for classic porphyrin´s PSs, attention is also focused on non-porphyrin photosensitizers 

such as anthraquinones, anthrapyrazols, xanthines, acridines or cyanines (Diwu and 

Lown, 1994). 

Recently, our in vitro experiments have showed that one group of acridine derivatives 

– 1’,1’’-(acridine-3,6-diyl)-3’,3’’-dialkyldithiourea hydrochlorides (AcrDTUs) generated 

oxygen free radicals after irradiation, what is one of the basic properties of each PS. 

In the present study, photocytotoxic effect of AcrDTU on a resistant line A2780/CP has 

been evaluated and compared with the effect on parental cell line A2780. 

The analysis of the photocytotoxicity of AcrDTUs showed (viability of ovarian cancer 

cell lines was monitored by MTT assay) that their effect was comparable for sensitive 

and resistant cells lines. After irradiation (365 nm, 1.05 J/cm2), propyl-AcrDTU (5 µM) 

decreased viability of sensitive and resistant cells about 95 % and 90 %, respectively. 

The photocytotoxicity was accompanied with changes of cell morphology. When cells 

were incubated with 0.5 µM propyl-AcrDTU and irradiated (365 nm, 1.05 J/cm2), the 

lost of typical cellular shape and shrinkage of cells was observed (2 h-treatment). Such 

changes of cytomorphology are typical hallmarks of apoptosis. Furthermore, generation 

of “DNA-ladder” (oligonucleosomal double-strand DNA fragments) has been confirmed 

after irradiation (365 nm, 1.05 J/cm2) and incubation of sensitive and resistant cell lines 

with 0.5 µM propyl-AcrDTU for 4 h. Cell cycle analysis (flow cytometry) revealed the 

accumulation of A2780 cells at the subG0 phase in a dose-dependent manner. 

To reveal the mechanism of apoptosis, the intracellular localization of AcrDTU derivatives 

in the A2780 and A2780/CP cell lines was monitored (ImageStream Imaging Flow 

Cytometer). 


This work was supported by STU Grant for Young Researchers 1327. 


References 

Diwu Z., Lown J.W.: Phototherapeutic potential of alternative photosensitizers to 

porphyrins. – Pharmacol Ther. 63, 1-35, 1994. 

Robertson C.A., Hawkins Evans D., Abrahamse H.: Photodynamic therapy (PDT): A short 

review on cellular mechanisms and cancer research applications for PDT. – J Photochem 

Photobiol B. 96, 1-8, 2009. 

P80. HAEMATOPOIESIS IN VITRO UNDER HIF-1 MODULATION 

Markéta Hanáčková 1,2 , Kateřina Štefková 1 , Lukáš Kubala 1,2,3 , Jiří Pacherník 1,3 

1 

Institute of Experimental Biology, Faculty of Science, Masaryk University, 

Kotlarska 2, 611 37 Brno, Czech Republic, 17059@mail.muni.cz 

2 

Institute of Biophysics, Academy of Sciences of the Czech Republic v. v. i., 

Kralovopolska 135, 612 65 Brno, Czech Republic 

3 


Engineering, St. Anne‘s University Hospital Brno, Brno, Czech Republic 

Hypoxia through modulation of hypoxia inducible factors (HIF) controls induction and 

progression of haematopoiesis. Here we focus on the role of HIF family member HIF-1α 

in regulation of haematopoiesis from embryonic stem cells. 

We have observed that depletion of HIF-1α accelerates haematopoiesis in embryonic 

stem cells in vitro. In mutated cells both erythroid progenitors (BFU-E and CFU-E) appear 

earlier and expression of prohaematopoietic genes is increased compared to wild-type 

cells. 

Recently it has been observed that deferoxamine (DFO) and other Fe ions chelators not 

only induce differentiation but also shift myeloid differentiation to monocyte lineage 

in acute myeloid leukaemia (Callens et al., 2010). DFO through Fe ion chelation also 

stabilizes HIF-1. Therefore we tested the effect of classical HIF stabilizing drugs (CoCl 2 

and DMOG) on adult and fetal haematopoiesis in vitro. 

In accordance with the effect of HIF-1α depletion, addition of CoCl 2 

to the culture 

reduced erythropoiesis (decreased number of BFU-E and CFU-E) in both adult (bone 

marrow-derived) and fetal (fetal liver-derived) hematopoietic progenitors. Interestingly, 

CoCl 2 

also decreased CFU-G together with CFU-GM but increased CFU-M number in fetal 

haematopoiesis in the same manner as DFO in acute myeloid leukemia study (Callens 

et al., 2010). In contrast, in adult hematopoietic progenitors CoCl 2 

increased number of 

CFU-G and CFU-GM but decreased CFU-M. 

In summary, our results show HIF-1α dependent regulation of both overall 

haematopoiesis and myeloid lineage directed differentiation. Moreover, the results 

of myeloid differentiation in response to CoCl 2 

in adult and fetal haematopoiesis may 

indirectly support recent hypothesis, that tumor cells may have the phenotype of fetal 

progenitors rather than the phenotype of adult lineage progenitors. 







CZ.1.05/1.1.00/02.0123). 

References 

Callens C, Coulon S, Naudin J, Radford-Weiss I, Boissel N, Raffoux E, Wang PH, Agarwal 

S, Tamouza H, Paubelle E, Asnafi V, Ribeil JA, Dessen P, Canioni D, Chandesris O, Rubio 

MT, Beaumont C, Benhamou M, Dombret H, Macintyre E, Monteiro RC, Moura IC, 

Hermine O.: Targeting iron homeostasis induces cellular differentiation and synergizes 

with differentiating agents in acute myeloid leukemia. - J Exp Med. 207(4):731-50, 2010 

P81. SULFORAPHANE UPREGULATES CD44, CD105 AND CD90 EXPRESSION IN MSCS 

PRESENT IN BONE MARROW 

Hunakova L. 1 , Altanerova V. 2 , Sulikova G. 1 , Kaiserova K. 2 , Altaner C. 1 

1 

Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic 

2 

St. Elisabeth Oncological Institute, Bratislava, Slovak Republic 

It has been accepted that mesenchymal (stromal) stem cells (MSCs) present in bone 

marrow mononuclear cells concentrate are therapeutic cells involved in regenerative 

process. Emerging evidence suggest that most of the beneficial effects of MSCs could be 

explained by secretion of soluble factors contributing by paracrine fashion to the multiple 

effects including modulation of inflammatory and immune reactions, modulation of cell 

death and stimulation of endogenous progenitor cells. 

Sulforaphane (SFN) is one of naturally occurring cancer chemopreventive isothiocyanates 

found in cruciferous vegetables, consumption of which has been associated with reduced 

risk of cancer. We found that low concentrations of SFN that do not affect the viability of 

bone marrow-derived MSCs as shown by MTT assay, increase the surface expression of 

CD105, CD44 and CD90 proteins. Recently, CD44 and CD90 expression have been shown 

to be associated with the responsiveness of CLI patients to bone marrow mononuclear 

cell therapy. 

Thus SFN might enhance the quality of MSCs as reflected on the expression of cell 

surface markers and find utilization in regenerative medicine. 


Financial support from the Slovak Grant Agency VEGA (grant 2/0177/11) and Slovak 

League against Cancer is gratefully acknowledged. 


P82. FLOW CYTOMETRIC ANALYSIS AND MULTIPLE LINEAGE DIFFERENTIATION OF 

ADIPOSE TISSUE-DERIVED STEM CELLS OF DIABETIC PATIENTS 

Kollárová Z. 1,2 Kubinová Š. 1,2 , Turnovcová K. 1,2 , Syková E. 1 

kollarova@biomed.cas.cz 

1 

Institute of Experimental Medicine ASCR, Prague, Czech Republic 

2 

2 nd Faculty of Medicine, Charles University, Prague, Czech Republic 

INTRODUCTION 

Adipose tissue is an abundant source of autologous adult stem cells that may open 

new therapeutic perspectives for the treatment of type I. and type II. diabetes and 

their complications. However, it is unclear whether the mesenchymal stem cells of 

diabetic patients, constantly influenced by hyperglycaemia, have the same properties as 

mesenchymal stem cells from non-diabetic patients. To study the differencies between 

adipose tissue-derived stem cells (ATSCs) isolated from diabetic and non-diabetic 

patients, cell surface markers were analyzed by flow cytometry and multiple lineage 

differentiation and the expression of adipose and osteogenic specific genes were studied. 

MATERIAL AND METHODS 

ATSCs from patients with type II. diabetes (n=6) and type I. diabetes (n=4) were compared 

to ATSCs from non-diabetic patients. The tissue samples were processed by enzymatic 

digestion, and cells were cultivated in full cultivation media containing MesenCult 

(Stemcell technologies, Vancouver, Canada) supplemented with 10% fetal bovine serum 

(PAA Laboratories, Coelbe, Germany), and Penicilin/Streptomycin (200 µg/ml; Lonza, 

Cologne, Germany). Cells from the third passage were characterized by their expression 

of surface markers using fluorescence-activated cell sorting (FACS). A total of 2x10 6 cells 

were resuspended in 1ml PBS and incubated with their appropriate antibodies for 20 

minutes at room temperature. Antibodies against human antigens CD31, CD34, CD45, 

CD29, CD105, CD73, CD90, CD235a, CD271, HLA-ABC, HLA-DR+DP, VEGFR2, CD146 and 

antifibroblast were used. Analysis of surface markers was performed on a FACSAria TM 

Flow Cytometer and their expression was evaluated on FACSDiVa Software. 

Standard protocols to differentiate the ATSCs into adipocytes, osteoblasts and 

chondrocytes were followed to confirm the cells’ multipotency. Cell differentiation into 

various lineages was confirmed by specific staining and real-time q-PCR. The expression 

of ACTB, GAPDH, ALPL, PPAR6, Runx2and LPL was analyzed on a real-time PCR System 

StepOnePlus (Applied Biosystems, San Francisco, CA,USA). 

RESULTS 

Flow cytometry revealed the expression of CD29, CD73, CD90, HLA-ABC and fibroblast 

markers and no expression of CD31, CD34, CD45, CD235a, CD271 or HLA-DR+DP was 

found in ATSCs from either diabetic or non-diabetic patients. Also, adipose differention 

potential was well-preserved in both groups of cells. However, in diabetic patients the 

osteogenic differentiation potential of ATSCs was decreased, which was confirmed by 

specific staining for Alizarin red and the gene expression of Runx2 and LPL. 

Grant support: GA ČR P304/11/0653, GA ČR 9304/11/0731, GA ČR P304/11/P633 


P83. DIFFERENTIATION OF NEURAL CREST CELLS FROM HUMAN EMBRYONIC STEM 

CELLS AND THEIR ODONTOGENIC INDUCTION 

Jan Křivánek 1 , Karel Souček 2,3 , Radek Fedr 2 , Eva Švandová 4 , Eva Matalová 4,5 , Aleš 

Hampl 1,3 

1 

Department of Histology and Embryology, Faculty of Medicine, Masaryk University, 


2 


Republic, v.v.i. 

3 


St. Anne’s University Hospital, Brno, Czech Republic 

4 

Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech 

Republic, v.v.i. 

5 

Department of Physiology, University of Veterinary and Pharmaceutical Sciences, Brno, 


Neural crest cells (NCC) are transient cell type which differentiates from invaginating 

neural plate when two opposite neural folds merge together. These cells are able to 

undergo an epithelial-mesenchymal transition, migrate through whole vertebrate 

embryo and form many terminal differentiated cell types such as melanocytes, 

odontoblasts, Schwann cells, connective tissue cells and many others. From this point 

of view NCC represent attractive cell type than can be used in regenerative medicine. 

Here we have differentiated human embryonic stem cells (hESCs) into cells possessing 

molecular and functional properties of NCC. The key step in the differentiation protocol 

was dual inhibition of SMAD signaling in its initial phase followed with FACS-mediated 

purification of CD271-positive cells at day 11 of differentiation. The obtained cells were 

found, by flow cytometry and immunocytochemistry, to express molecular markers 

characteristic of NCC such as HNK1, Sox9, Sox10, and AP2αβ. Importantly, these putative 

NCC are also capable of in vitro differentiation into adipogenic cell lineage. When 

exposed in vitro to FGF8 these putative NCC up-regulate expression of homeobox genes 

that are normally produced early in odontogenesis. Currently, we are investigating the 

developmental potential of hESC-derived NCC using chick embryos and also we are 

testing the stability of molecular and functional phenotype of these cells when they are 

long-term propagated in our modified culture media on dishes coated by poly-L-ornithin 

and fibronectin. 

This work was supported by: CZ.1.07/2.3.00/20.0185, GAP304/11/1418, 

MUNI/A/0962/2012, CZ.1.05/1.1.00/02.0123 and MSM0021622430. 


P84. THE ROLE OF ATP-BINDING TRANSPORTERS ASSOCIATED WITH MULTI-DRUG 

RESISTANCE IN STEM CELLS 

Martina Lánová 1 , Josef Večeřa 1 , Jan Kučera 1 , Jiřina Procházková 1 , Jiří Pacherník 1,2 

1 


Republic 

2 


Engineering, St. Anne’s University Hospital Brno, Czech Republic 

ATP-binding transporters (ABC-t) play various roles in regulation organism function and 

homeostasis from prokaryota to mammals. ABC-t mediate transport of mainly lipophilic 

substances through cellular membranes. Some ABC-t are important in cell protection 

against endogenous and importantly also exogenous toxins. These transporters are 

called ABC-t associated with multi-drug resistance (ABC-t/MDR), according to their role 

in resistance of tumor cells to pharmacotherapy. ABC-t/MDR are also over-expressed in 

stem cells, where their protective role is expected, too. Particularly, ABCB1, ABCC1, and 

ABCG2 are common ABC-t/MDR expressed in stem cells. However, substrates of ABC-t/ 

MDR are not only toxins, but also important signaling molecules as well leukotrienes 

and/or glutathione conjugates and porphyrins, which mediated balance in intracellular 

oxidation-reduction processing. Thus we hypothesize the role of ABC-t/MDR also in 

regulation of stem cells fate. To test this hypothesis we analyzed effect of modulation of 

ABC-t/MDR activity in embryonic and neural stem cells. We observed that ABCC1 and 

ABCG2 are the most expressed ABC-t/MDR in our tested stem cells. Importantly, inhibition 

of these ABC-t/MDR leads to decreasing of stemness and induction of differentiation in 

both embryonic and neural stem cells. Analysis of mechanism of observed effect and 

identification of studying ABC-t/MDR substrates, which may be responsible for this 

effect, are in progression. 

Acknowledgment 

This work was supported by grant from MEYS CR Cost CZ LD11015 and Ministry of 

Education, Youth and Sport of the Czech Republic MSM0021622430 

P85. CYTOKINE SECRETION DURING NEURAL PROGENITOR CELL DIFFERENTIATION 

Karla Jarkovska 1 , Katerina Mairychova 1 , Rita Hrabakova 1 , Jirina Tyleckova 1 , Martin 

Marsala 2 , Silvia Marsala 2 and Hana Kovarova 1 

1 

Institute of Animal Physiology and Genetics, AS CR v.v.i., Libechov, Czech Republic 

2 

University of California, San Diego, United States of America 

Neural stem cells have an enormous potential in the therapy of the neurodegenerative 

diseases or spinal cord injuries. For therapeutic applications it is essential to characterise 

the developmental stages of neural cells from proliferating progenitors to fully 

differentiated neural cells. However, suitable biomarkers indicating commitment to a 


given cell type are missing and these may facilitate the selection of optimal clones for 

cell transplantation 

In addition, the demands on treatment of neurodegenerative diseases are expected to 

rise due to the increase in long-aged human population and higher disease incidence. 

During the last two decades, stem cells discovery revolutionised the search for new 

therapies and possible neural stem cells transplantation is a focus for many researchers, 

some of them reporting significant function improvement after transplantation (Lu et 

al., 2012). The aim of stem cells characterisation is, inter alia, to identify molecules 

monitoring post-transplantation stages in the course of cell-based therapies. The 

proteins secreted by cells, called secretome, offer such opportunities, hence the aim 

of this study is characterisation of secretome of neural progenitor cells and its changes 

during neural differentiation. 

The human neural stem cells derived from foetal spinal cord were isolated by FACS 

according to described cell surface signatures from heterogeneous population (Yuan 

et al., 2011). Sorted cells were cultivated in monolayer by bFGF and subsequently 

differentiated by adding BDNF and GDNF for 30 days. Media were collected every other 

day of neuronal differentiation. For the quantification of the cytokines level we selected 

the Luminex xMAP approach followed by statistical evaluation and interaction network 

modeling. The most significant quantitative changes were observed in the level of 10 

cytokines (CCL2, CCL3, CCL5, CXCL1, CXCL5, CXCL8, IL-10, MCSF, VEGF, angiogenin) with 

three different cytokine secretion profiles during differentiation. 

Our findings suggest a set of new cytokines which could help in monitoring of precursor 

cells differentiation and survival as well as therapeutic effect. Despite that, further 

studies on stem cells secreted proteins are needed. 


This study was supported by Technical Agency of the Czech Republic (TA01011466) and 

the project ExAM from European Regional Development Fund CZ.1.05/2.1.00/03.0124. 

References 

Lu, P., Wang, Y., Graham, L., McHale, K., Gao, M., Wu, D., Brock, J., Blesch, A., Rosenzweig, 

E.S., Havton, L.A., et al. (2012). Long-Distance Growth and Connectivity of Neural Stem 

Cells after Severe Spinal Cord Injury. Cell 150, 1264–1273. 

Yuan, S.H., Martin, J., Elia, J., Flippin, J., Paramban, R.I., Hefferan, M.P., Vidal, J.G., Mu, Y., 

Killian, R.L., Israel, M.A., et al. (2011). Cell-Surface Marker Signatures for the Isolation of 

Neural Stem Cells, Glia and Neurons Derived from Human Pluripotent Stem Cells. PLoS 

ONE 6, e17540. 

Corresponding author: 

Katerina Mairychova, katerina.mairychova@gmail.com 


P86. THE MECHANISM OF RESPONSE TO HYPOXIA IN EMBRYONIC STEM CELLS 

Julie Musilová 1 , Jan Kučera 1 , Martina Lánová 1 , Kateřina Štefková 1 , Lukáš Kubala 1,2,3 , Jiří 

Pacherník 1,2 

1 


Republic; 356419@mail.muni.cz 

2 

International Clinical Research Center – Centre of Biomolecular and Cellular; 

Engineering, St. Anne’s University Hospital Brno, Czech Republic 

3 

Institute of Biophysics ASCR v.v.i., Brno, Czech Republic 

Oxygen is a crucial element for life of most organisms. Key regulator of oxygen sensing 

and responding intracellular machinery is heterodimeric transcription factor HIF1 

(hypoxia inducible factor 1). Presence of oxygen leads to rapid ubiquitination and 

proteozomal degradation of cytoplasmic subunit HIF1α. This process is abolished in 

hypoxia, leading to accumulation of α subunit and dimerization with HIF1β (also called 

ARNT). Upon binding of HIF1 heterodimer to hypoxia responsive elements, transcription 

of many specific genes involved in oxygen homeostasis is induced (Wang et al., 1995). 

Moreover hypoxia increases a production of intracellular reactive oxygen species (ROS) 

(Chandel et al., 2002). ROS can modulate cell signaling through transcription and also 

through posttranslational modifications of intracellular signaling pathways (Finkel, 2011). 

Additionally, further mechanisms of low oxygen sensing excluding the involvement of 

HIF and ROS were reported (Wouters and Koritzinsky, 2008). Thus, so called hypoxia 

signaling could be promoted via different HIF and ROS dependent and independent 

manners. Since hypoxia is known to regulate stem cell fate both in vivo and in vitro, 

the main aim of our study is to clarify the role of HIF and ROS in hypoxic response of 

embryonic stem cell. 

We used wild-type and HIF1α deficient (-/-) mouse embryonic stem cell (mES) to analyze 

signaling pathways responsible for stemness maintenance. We compared effects 

of hypoxia (1% O 2 

) and pharmacologically induced stabilization of HIF in normoxic 

conditions on mES signaling. To clarify effects of ROS, various redox modulators were 

involved in our study. Posttranslational modifications of JAK/STAT3, MEK/ERK, PI3K/ 

Akt, p38 and Notch signaling cascades were analyzed, as well as effects on proliferation, 

differentiation and apoptosis these mES cells. 

Our data show HIF1α and ROS-dependent down regulation of MEK/ERK signaling under 

hypoxic conditions. On the other hand, either hypoxia or ROS scavengers do not affect 

activation of JAK/STAT3 signaling. Activities of PI3K/Akt and p38 signaling pathways 

were downregulated independently on HIF1α or ROS. Interestingly, the pharmacological 

inhibition of prolylhydroxylases, which leads to increase of HIF1 stability in normoxia, 

does not affect any analyzed signaling pathways. 

In conclusion, our results clearly document the complexity of cellular responses to 

hypoxic conditions, where the important part of such responses is HIF1α independent. 


This work was supported by grants from MEYS CR MSM0021622430 and Cost CZ LD11015, 

and from the European Social Fund - projects OganoNET (CZ.1.07/2.4.00/31.0245) 


and HistoPARK (CZ.1.07/2.3.00/20.0185). LK was supported by the European Regional 

Development Fund - Project FNUSA-ICRC (No. CZ.1.05/1.1.00/02.0123). 

References 

Chandel, N.S., McClintock, D.S., Feliciano, C.E., Wood, T.M., Melendez, A., Rodriguez, 

A.M., Schumacker, P.T.: Reactive oxygen species generated at mitochondrial complex III 

stabilize hypoxia-inducible factor-1α during hypoxia: a mechanism of O2 sensing. – J Biol 

Chem 275: 25130-25138, 2000. 

Finkel, T.: Signal transduction by reactive oxygen species. – J Cell Biol 194 (1): 7-15, 2011. 

Wang G. L., Jiang B. H., Rue E. A., Semenza G. L.: Hypoxia-inducible factor 1 is a basichelix-loop-helix-PAS 

heterodimer regulated by cellular O2 tension. – Proc Natl Acad Sci 

USA 92: 5510–5514, 1995. 

Wouters, B. G. and Koritzinsky, M.: Hypoxia signalling through mTOR and the unfolded 

protein response in cancer.: – Nat Rev Cancer. 8: 851-64, 2008. 

P87. ROLE OF HIF-1Α IN SELFRENEW AND DIFFERENTIATION OF NEUROSPHERES 

DERIVED FROM EMBRYONIC STEM CELLS 

Pánská V. 1 , Večeřa J. 1 , Kubala L. 2,3 , Pacherník J. 1,3 

1 


Republic; veronika.panska@centrum.cz 

2 


3 

ICRC - CBCE, FNUSA, Brno, Czech Republic 

Hypoxia inducible factor 1α (HIF-1α) is a main factor which responds to hypoxia 

and regulates adaptation to hypoxic conditions. HIF-1α is important for stemness 

maintenance by stabilizing activated Notch-1 (NICD) and for maintenance of neurogenic 

niche by upregulating vascular endothelial grow factor (VEGF) (Gustafsson et al., 2005; 

Covello et al., 2004). 

These informations are important for our experiments with neural stem cells (NSC). We 

use NSCs derived from murine wt and HIF-1α deficient embryonic stem cells (ES) and 

NSCs dissected from embryonic brain (Hitoshi et al., 2004). We monitor influence of 

HIF-1α knock-out on formation, selfrenewal capacity and transition ability of NSCs by 

neurosphere cultivation (colony forming assay) and by screening of neural markers using 

qRT-PCR and WB techniques. 

We also use flow cytometry for confirmation of neural attributes of our NSCs and for 

quantifying cells with neural characteristics. In this assay we work with neural markers 

such as Forse-1 and Pax-6. 

According to our preliminary results from qRT-PCR and colony forming assay, HIF-1α 

supports selfrenewal of NSCs and knockout of HIF-1α enables neurospheres to express 

more neural markers. Moreover, gene expression of Hes1 and Hes5 transcription factors 

is affected by HIF-1α knockout which poits to interaction of HIF-1α and Notch pathways 

in selfrenewal abilities of NSC. 



This work was supported by grants from MEYS CR MSM0021622430 and Cost CZ LD11015 

and from the European Social Fund - projects OganoNET (CZ.1.07/2.4.00/31.0245) 

and HistoPARK (CZ.1.07/2.3.00/20.0185). LK was supported by the European Regional 

Development Fund - Project FNUSA-ICRC (No. CZ.1.05/1.1.00/02.0123). 

References 

Covello KL, and Simon MC: HIFs, hypoxia, and vascular development. - Curr Top Dev Biol 

62:37–54, 2004. 

Gustafsson MV, Zheng X, Pereira T, Gradin K, Jin S, Lundkvist J, Ruas JL, Poellinger 

L, Lendahl U, and Bondesson M: Hypoxia requires notch signaling to maintain the 

undifferentiated cell state. - Dev Cell 9:617–28, 2005. 

Hitoshi S, Seaberg RM, Koscik C, Alexson T, Kusunoki S, Kanazawa I, Tsuji S, and van 

der Kooy D: Primitive neural stem cells from the mammalian epiblast differentiate to 

definitive neural stem cells under the control of Notch signaling. - Gene Dev 18:1806– 

11, 2004. 

P88. HEMATOPOIESIS OF MOUSE EMBRYONIC STEM CELLS – THE ROLE OF P38ALPHA 

KINASE 

Kateřina Štefková 1 , Markéta Hanáčková 1,3 , Jan Kučera 1 , Lukáš Kubala 1,2,3 , Jiří Pacherník 1,2 

1 


Republic, stefkova.katka@gmail.com 

2 


Engineering, St. Anne‘s University Hospital Brno, Czech Republic 

3 


Hematopoietic differentiation of embryonic stem cells (ESCs) in vitro has been used as a 

model to study early hematopoietic development. Identifying the molecular pathways 

regulating hematopoietic stem cell (HSC) or hematopoietic progenitor cell (HPC) 

specification, self-renewal, and expansion remains a fundamental goal of both basic and 

clinical biology. 

Similarly to the other members of MAPK family, p38 signaling is involved in plenty 

of cell responses from cell cycle to apoptosis, induction of cytokine genes, and 

differentiation. Recently, it has been shown that MAPK p38α plays a role in regulation of 

adult hematopoiesis, particularly erythropoiesis and granulopoiesis (Geest C.R., Coffer 

P.J., 2009). Besides MAPK signaling has been demonstrated to play a key role in the 

maintenance of HSC quiescence (Ito K. et al, 2006). 

The aim of our work was to study the mechanisms and effectiveness of hematopoiesis 

in mouse ES cells, in consideration of mitogen-activated protein kinase (MAPK) p38. 

We suppose that if p38α kinase participates on the maintenance of the hematopoietic 

stem and early progenitor cells, depletion of p38α kinase changes the dynamics of 

hematopoiesis in p38α -/- when compared to the wildtype p38 ES cell line. So during 

differentiation of mentioned ES cells we determined dynamic of hematopoiesis using 

qRT-PCR, colony-forming assay and FACS phenotyping. 


Our results show that both p38α +/+ and p38α -/- cells are able to be directed to 

hematopoietic cell lineages. However p38α deficient cells undergo hemasopoiesis more 

intensively in contrast to their wild-type counterpart. P38α -/- cells also in higher ratio 

form erythroid and granulopoietic lineages. In contrast, wild-type cells are able to better 

maintain CD34 positive cell sub-population. Thus based on our preliminary data we can 

hypothesize that depletion of p38α MAPK leads both to attenuating stemness and to 

enhancing myeloid progenitor´s development. 






CZ.1.05/1.1.00/02.0123). 

References 

Geest CR. Coffer PJ.: MAPK signaling pathways in the regulation of hematopoiesis., J 

Leukoc Biol. 86, 237–250, 2009 

Ito, K., Hirao, A., Arai, F., Takubo, K., Matsuoka, S., Miyamoto, K., Ohmura, M., Naka, K., 

Hosokawa, K., Ikeda, Y., Suda, T.: Reactive oxygen species act through p38 MAPK to limit 

the lifespan of hematopoietic stem cells., Nat. Med. 12, 446–451, 2006 

P89. HYPERICIN SUPPRESSES “SIDE POPULATIONS” BUT STIMULATES AGGRESSIVE 

PHENOTYPE IN A549 HUMAN LUNG ADENOCARCINOMA CELLS 

Jana Vargová, Jaromír Mikeš, Lucia Mikešová, Zuzana Zhihovicsová, Peter Fedoročko 


Košice, Slovak Republic; jana.vargova1@student.upjs.sk 

Causes of tumor resistance to therapy and/or relapses are of major interest in cancer 

research, recently. These issues represent the most serious aspects of cancer. In the 

light of the latest findings, they are being linked with existence of small fraction of 

cells resembling stem cells with their nature, called cancer stem-like cells (CSC). One 

of methods for detection of CSC is the so called “Side population” (SP) technique. It 

is based on ability of certain cells to get rid of xenobiotics, including fluorescent dye 

Hoechst 33342. From molecular point of view, this phenomenon is associated with 

elevated expression of ABC-transporter molecules, above all ABCG2 (BCRP) (Zhou et al, 

2001). 

Hypericin (HYP) is compound commonly found in St. John’s Wort (Hypericum perforatum). 

HYP has been widely used in combination with photodynamic therapy. However, it has 

been shown that HYP possess some antitumor activity in the dark, too (Blank et al., 

2004). HYP was suggested as a potential substrate of ABC-molecules, mostly MRP1 and 

BCRP. Moreover, HYP per se stimulated expression of MRP1 and BCRP in HT-29 cells 

(Jendzelovsky et al., 2009). 

The aim of our study was to explore the effect of non-photoactivated HYP on 


SPsubpopulation occurrence and its nature in human lung adenocarcinoma cells 

A549. We hypothesized that HYP could act as enhancer of SP phenotype and stimulate 

the aggressive nature of these cells, as the result of promotion of ABC-transporters 

expression. 

We demonstrated strong correlation between higher activity of ABC transporters in 

SP cells and HYP elimination. It supports an idea of HYP being the substrate for ABCtransporters 

as suggested by Jendzelovsky and colleagues (Jendzelovky et al., 2009). 

On the other hand, however, we found that HYP reversibly suppresses SP population 

in dose-dependent manner. To assess the influence of HYP on the clonogenic capacity 

of A549 cells, we chose two methodologies, i.e. standard assay and single cell cloning. 

We observed certain paradox, when HYP enforced the clonogenicity of A549 when 

established by standard test, in contrary, though, we saw lower clonogenic efficiency but 

larger colonies established by single cell cloning. 

Our results demonstrate the influence of non-photoactivated HYP on suppression of 

Side population in reversible manner. We suggest, that this effect could by caused via 

one of known pathways involved in effect of HYP in dark which include affection of 

key targets in CSC, e.g. enhanced ubiquitinylation of Hsp90 (Blank et al., 2003; Peng et 

al., 2007). HYP was also capable to induce more aggressive phenotype in A549 cells. 

We conclude that differences in observed clonogenic capability could be explained by 

elevated dependence of cells with more aggressive phenotype on factors produced by 

surrounding cells. Further studies will be necessary to confirm this suggestion and to 

assess the impact of “niché – CSC“ relation on their behavior. 



contract No. APVV-0040-10 and VVCE-0001-07 and the Scientific Grant Agency of the 


References 

Blank, M., Mandel, M., Keisari, Y., Meruelo, D. and Lavie, G.: Enhanced ubiquitinylation 

of heat shock protein 90 as a potential mechanism for mitotic cell death in cancer cells 

induced with hypericin. – Cancer research 63: 8241-8247, 2003. 

Blank, M., Lavie, G., Mandel, M., Hazan, S., Orenstein, A., Meruelo, D. and Keisari, Y.: 

Antimetastatic activity of the photodynamic agent hypericin in the dark. – International 

journal of cancer 111: 596-603, 2004. 

Jendzelovský, R., Mikes, J., Koval‘, J., Soucek, K., Procházková, J., Kello, M., Sacková, 

V., Hofmanová, J., Kozubík, A. and Fedorocko, P.: Drug efflux transporters, MRP1 and 

BCRP, affect the outcome of hypericin-mediated photodynamic therapy in HT-29 

adenocarcinoma cells. – Photochemical & Photobiological Sciences 12: 1716-1723, 2009. 

Peng, C., Brain, J., Hu, Y., Goodrich, A., Kong, L., Grayzel, D., Pak, R., Read, M. and Li, 

S.: Inhibition of heat shock protein 90 prolongs survival of mice with BCR-ABL-T315Iinduced 

leukemia and suppresses leukemic stem cells. – Blood 110: 678-685, 2007. 

Zhou, S., Schuetz, J.D., Bunting, K.D., Colapietro, A.M., Sampath, J., Morris, J.J., Lagutina, 

I., Grosveld, G.C., Osawa, M., Nakauchi, H. and Sorrentino, B.P.: The ABC transporter 

Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant 

of the side-population phenotype. – Nature Medicine 7:1028-1034, 2001. 


P90. TOLL-LIKE RECEPTOR 2 TRACKS THE EMERGENCE OF EARLIEST MYELOID 

PROGENITORS IN PRECIRCULATION EMBRYO 

Jana Balounová, Tereza Vavrochová, Martina Benešová and Dominik Filipp 

Institute of Molecular Genetics, AS CR, Prague, Czech Republic; 

jana.balounova@img.cas.cz 

Toll like receptors (Tlrs) are critically important in the pathogen recognition and 

regulation of the innate and adaptive immune responses (Akira et al., 2001; Medzhitov, 

2007). In addition, a direct pathogen sensing of bone marrow hematopoietic stem 

cells and hematopoietic progenitors via Tlrs seem to play a crucial role in directing the 

hematopoietic cell fates under inflammatory conditions (De Luca et al., 2009; Nagai et 

al., 2006; Sioud et al., 2006). 

So far the expression of Tlrs during early stages of embryonic development has not 

been addressed. Using a transgenic model to trace cells of embryonic origin we show 

that Tlrs are expressed on embryonic myeloid cells as well as hematopoietic precursors 

committed to the myeloid lineage. Together with the prototypic marker of hematopoietic 

progenitors, c-kit, TLR2 is specifically expressed on the surface of hematopoietic 

precursors in early gastrulation embryos (E6.5-E7.0). E7.5-E8.5 TLR2 + c-kit + cells express 

CD45 mRNA and gradually differentiate through an intermediate c-kit + CD45 + stage to 

c-kit – CD45 + myeloid cells. Moreover E6.5 TLR2 + precursors differentiate to myeloid TLR2 + 

ckit – CD45 + cells in vitro. Upon TLR2 ligand recognition, E8.5 TLR2 + c-kit + cells proliferate 

and differentiate to CD45 + CD11b + myeloid cells in a MyD88 dependent manner. 

Our results indicate that TLR2 could be used as one of the earliest markers of 

embryonic hematopoietic progenitors and suggest a functional link between embryonic 

hematopoiesis and inflammation. 


This study was supported by the Grant Agency of the Academy of Sciences of the Czech 

Republic (grant IAA500520707) and by The Grant Agency of Charles University (grant 

259109). 

References 

Akira S, Takeda K, Kaisho T. 2001. Nat Immunol 2: 675-680. 

De Luca K, et al. 2009. Leukemia 23: 2063-2074. 

Medzhitov R. 2007. Nature 449: 819-826. 

Nagai Y, et al. 2006. Immunity: 24(6): 801-812 

Sioud M, et al. 2006. Journal of Molecular Biology 364(5): 945-954 


P91. PHENOTYPE AND ACTIVATION STATE OF T AND NK CELLS IN PORCINE 

COLOSTRUM 

Karolina Hlavova, Hana Stepanova, Martin Faldyna 

Department of Immunology, Veterinary Research Institute, Brno, Czech Republic; 

hlavova@vri.cz 

The distribution and activation markers of T cell subpopulations and NK cells in porcine 

colostrum were studied. The results indicate that the absolute number of lymphocytes 

in colostrum is the highest in the first day of lactation. The most predominant 

subpopulation were cytotoxic T cells followed by CD4+CD8+ double positive T cells, 

CD2+CD8+ γδ T cells and NK cells. Helper T cells and other γδ T cell subpopulations 

were present in colostrum in very low percentages. The study of the distribution of 

T cell subpopulations during the first 3 days of lactation revealed that the proportion 

remains constant, but the absolute numbers of T- and NK cells decrease significantly 

in the first hours of lactation. The expression of CCR7, CD11b, CD25, CD45RA and 

MHCII-DR was used to detect the activation state of T- and NK cells in colostrum. T-cell 

subpopulations showed central/effector memory phenotype suggesting that these are 

antigen experienced cells. NK cells showed high frequency of MHCII-DR+ cells. 


The study was supported by the Ministry of Agriculture of the Czech Republic 

(MZE0002716202) and project AdmireVet (CZ 1.05/2.1.00/01.0006; ED0006/01/01). 

P92. DISTRIBUTION AND FUNCTION OF PORCINE MONOCYTES IN DIFFERENT 

LYMPHOID TISSUE AND THE LUNG DURING EXPERIMENTAL ACTINOBACILLUS 

PLEUROPNEUMONIAE INFECTION 

Petra Ondrackova, Lenka Leva, Zdenka Kucerova, Monika Vicenova, Marketa 

Mensikova, Martin Faldyna 

Department of Immunology, Veterinary Research Institute, Brno, Czech Republic; 

ondrackovap@vri.cz 

Monocytes play an essential role in the defense against bacterial pathogens. Bone 

marrow (BM) and peripheral blood (PB) monocytes in pigs consist of two main „steadystate“ 

subpopulations: CD14 hi /CD163 - /SLA-DR - and CD14 low /CD163 + /SLA-DR + . During 

inflammation, the subpopulation of “inflammatory” monocytes expressing very high 

levels of CD163, but lacking SLADR molecule (being CD14 low /CD163 + /SLADR - ) appears in 

the BM and PB and replaces the CD14 low /CD163 + /SLADR + subpopulation. However, the 

current knowledge of monocyte migration into inflamed tissues in pigs is limited. 

The aim of the present study was to evaluate distribution of “inflammatory” CD14 low / 

CD163 + /SLADR - monocytes during experimental inflammation induced by Actinobacillus 

pleuropneumoniae (APP), ability of these cells to produce cytokines and a possible role 


for chemokines in attracting “inflammatory” CD14 low /CD163 + /SLADR - monocytes into the 

tissues. 

Monocyte subpopulations were detected by flow cytometry. Chemokines and chemokine 

receptors were detected by RT-qPCR. Cytokines were detected by flow cytometry, 

imunohistochemistry, immunofluorescence and RT-qPCR. 

The “steady-state” CD14 hi /CD163 - /SLA-DR - and CD14 low /CD163 + /SLA - monocytes were 

found in the BM, PB, spleen and lungs of control pigs. After APP-infection, “inflammatory” 

CD14 low /CD163 + /SLADR - monocytes replaced the “steady-state” subpopulation in BM, 

PB, spleen and they, moreover, appeared in an unaffected area, demarcation zone and 

necrotic area of the lungs and in tracheobronchial lymph nodes. They did not appear in 

mesenteric lymph nodes. 

Despite the systemic inflammatory reaction after APP infection, BM and PB 

“inflammatory” CD163 + monocytes did not express elevated levels of a wide range of 

cytokines compared to control pigs. The further analysis, however, showed that after in 

vitro stimulation with LPS from APP either BM or PB “inflammatory” CD163 + monocytes 

were able to produce significant amounts of pro-inflammatory cytokine TNF-α. This 

ability was, however, altered in APP-infected pigs. Finally, it was shown in APP-infected 

pigs that, contrary to BM and PB CD163 + monocytes, CD163 + monocytes infiltrating the 

necrotic area of inflamed lungs and tracheobronchial lymph nodes produced significant 

amounts of TNF-α, IL-1β, IL-6 and IL-8. 

Levels of mRNA for various chemokines with their appropriate receptors were found 

to be elevated in BM (CCL3-CCR1/CCR5, CCL8-CCR2/CCR5, CCL19-CCR7), necrotic area 

of lungs (CCL3-CCR1, CCL5-CCR1/CCR3, CCL11-CCR3, CCL22/CCR4) and tracheobronchial 

lymph nodes (CCL3-CCR1) and therefore they could play a role in attracting monocytes 

into inflamed tissues. 

In conclusion, “inflammatory” monocytes migrate from BM into PB and spleen, and 

further into the inflamed tissue and draining lymph nodes. Various chemokines could 

drive this migration. Moreover, PB and BM CD163 + monocytes do not contribute to the 

elevated cytokine levels in plasma during APP infection. On the other hand, CD163 + 

monocytes are source of pro-inflammatory cytokines in the lung lesions and draining 

lymph nodes during APP infection. 


This study was supported by grants of Czech Science Foundation (P502/10/P362), 

MZe 0002716202 and Ministry of Education, Youth and Sports of the Czech Republic 

(CZ.1.05/2.1.00/01.0006, AdmireVet). 


P93. FLOW CYTOMETRY STUDY OF IMMUNE RESPONSE AFTER ADMINISTRATION OF 

β-D-GLUCAN AND LOW DOSE OF T-2 TOXIN IN CHICKENS 

Viera Revajová 1 , Martin Levkut 1 , Viera Spišáková 1 , Zuzana Ševčíková 1 , Katarína 

Bobíková 1 , Eva Husáková 1 , Mária Levkutová 1 , Dominika Pejová 1 , Ema Paulovičová 2 , 

Mikuláš Levkut 1 

1 

University of Veterinary Medicine and Pharmacy, Slovak Republic; revajova@uvm.sk 

2 

Institute of Chemistry Slovak Academy of Science, Slovak Republic 

There is an increasing awareness of the hazard posed to both human and animal health 

by the presence of toxins produced by fungi in food and feed. Mycotoxins, where T-2 

toxin is included, have a diversity of chemical structures which accounts for different 

biological effects. They can be carcinogenic, mutagenic, teratogenic, immunotoxic, 

etc. One way of trying to inhibit the uptake of mycotoxins from contaminated feed is 

the use of mycotoxin binders. Facing the relative inefficacy of the clay binders towards 

mycotoxins others than aflatoxins, natural organic binders, such as yeast and lactic 

acid bacteria, have been shown to bind the different mycotoxins strongly to cell wall 

components (Kolossova et al., 2009). Another beneficial effects of lactic acid bacteria 

and yeasts are nutritional values and improving of immunity. 

A significant amount of ß-glucan entered the proximal intestine shortly after ingestion. 

Its transit through the proximal intestine decreased during time with a simultaneous 

increase in the ileum. Despite low systemic blood levels (less than 0.5%), significant 

systemic immunomodulating effects in terms of humoral and cellular immune responses 

were demonstrated (Větvička et al., 2007). Based on in vitro studies, ß-glucans act on 

several immune receptors including Dectin-1, complement receptor (CR3) and TLR-2/6 

and trigger a group of immune cells including macrophages, neutrophils, monocytes, 

natural killer cells and dendritic cells. As a consequence, both innate and adaptive 

response can be modulated by ß-glucans and they can also enhance opsonic and nonopsonic 

phagocytosis (Chan et al., 2009) and various types of interleukins. 

T-2 toxin was first isolated from the mould Fusarium tricinctum and belongs to nonmacrocyclic 

type A trichothecenes. After exposure by oral, dermal or inhalation route it 

can cause severe effects in various animal organs and tissues. In poultry, the toxic effects 

can be classified as genotoxic, cytotoxic, immunomodulatory.T-2 toxin can damage 

digestive system and liver, nervous system, skin and impairment poultry performance 

(Sokolović et al., 2008). Daily tolerated dose for T-2 toxin was established to 0.06 μg.kg -1 / 

body weight and limited dose in diet is 0.1 mg.kg -1 . 

T-2 toxin is a mycotoxin with immunomodulatory activity. Its mode of action is time- and 

dose-dependent. Immunosupression is the result of high doses. Immunostimulation is 

caused by low doses, and is evidenced by increase serum IgA and IgE antibodies because 

of rapid and transient activation of the genes responsible for the function of the immune 

system as well as genes important for inflammation response (Minervini et al., 2005). 

In the present study ß-D-glucan isolated from Candida albicans was perorally administered 

to chickens prior to feeding the diet artificially contaminated with low dose of T-2 toxin. 

The aim of the experiment was to determinate the changes of immunocompetent cells 


in immune organs (Bursa of Fabricii, spleen, tymus), intestine, and blood. To find out 

whether ß-D-glucan can induce immunologic response through T and B cell activation 

during T-2 toxin administration. For this purpose 4 group of chickens – control (C), 

ß-D-glucan (G), toxin (T) and combine ß-D-glucan+toxin (GT) – were investigated. 

Immunofenotyping of isolated lymphocytes by direct immunofluorescence method, 

double staining by mouse anti-chicken monoclonal antibody and flow cytometry for 

measuring of relative percentage were used. 

Determination of IgM+, IgG+ and IgA+ lymphocytes in Bursa of Fabricii showed increase 

(P

ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o.

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ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o.