Mezinárodní konference Analytical Cytometry V - pokračování

POSTERS

Specific practice in plasma cell separation from the bone marrow of monoclonal gammopathy of undetermined significance (MGUS) patients

Burešová I.¹, Muthu Raja K. R.¹, Kovářová L.^1,2, Perutka T.¹, Suská R.¹, Hájek R.^1,2,3

¹University research centre - Czech myeloma group (URC-CMG), Faculty of Medicine, Masaryk University Brno, Czech Republic; e-mail: buresova.i@seznam.cz

²Department of clinical hematology, LEHABI, Faculty Hospital Bohunice, Brno, Czech Republic

³Clinic of Internal Medicine – Hematology and Oncology, Faculty Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic

Monoclonal gammopathy of undetermined significance (MGUS) is asymptomatic plasma cell (PC) disorder which may progress into the symptomatic multiple myeloma (MM). One of the diagnostic criteria of MGUS is the presence of PC in BM below 10% (Rajkumar, 2005). The PC infiltration is higher than in healthy individuals (less than 2%) and lower than in MM patients. But these numbers are relevant for morphological evaluation of BM smears, in flow cytometry analysis the frequencies are lower (Rawstron et al., 2008). MGUS research cannot get along without plasma cell separation. In our laboratory, we have a FACSAria high-speed sorter from BD Biosciences, which can sort low frequency populations (0.2–5%) with high purity (over 90%). Nevertheless some types of samples demand to use specific practice.

Heparinized BM is aspirated after patient’s formal consent and immediately diluted with the same volume of Iscove’s medium. The mononuclear fraction is isolated by gradient centrifugation (Histopaque 1077). PCs are labeled by the antibody against CD138 (universal PC marker), conjugated with a fluorochrome.

Samples with very low infiltration (less than 0.2%) and low overall cellularity (less than 10 × 10⁶ cells): Such samples have very low yield of cells. When we sort plasma cells directly onto microscope slides, even limited amount of cells offer preparations on which FISH probes can be applied. Microscope slides are placed on the „ACDU holder“, which is the optional part of our sorter. Usually we sort 1000 cells onto one slide; we use „High“ sort setup and „Single cell precision mode“. The presence of cells can be verified under the microscope; cells are floating in a small drop of sheath fluid.

Samples with low infiltration and high overall cellularity (over 40 × 10⁶ cells): Sorting of such samples is extremely time consuming (2 or more hours of proper sorting) with the negative effects on cell viability. For such samples we use combination of magnetic and fluorescent separation. Fluorescently labeled, and washed sample is incubated with the antibody against the used fluorochrome, the antibody is conjugated with the magnetic particle. The cell suspension is than applied onto the LS column in the VarioMACS device and both negative (CD138^-) and positive (CD138⁺) fractions are collected. Positive fraction can be immediately sort to high purity on FACSAria.

Our results showed that BM samples from MGUS patients are specific by the low infiltration of target population and often high overall cellularity. Standard sort cannot always ensure good purity and good yield of target population and it is necessary to use less common practice or combined procedures for PC separation. Supplying of highly purified cells is the first condition for MGUS research on cellular and molecular level and offers new possibilities to our research teams.

This work was supported by the Ministry of Education grants LC06027 and MSM0021622434.

References

1. Rajkumar SV. MGUS and Smoldering Multiple Myeloma: Update on Pathogenesis, Natural History, and Management. Hematology Am Soc Hematol Educ Program 2005; 340–345.

2. Rawstron AC, et al. Report of the EMN on multiparametric FC in MM and related disorders. Haematologica 2008; 93: 431–438.

The use of seven-color flow cytometry for detection of mononuclear phagocyte subpopulations during salmonella infection in pigs

Zelníčková-Ondráčková P.^1,2, Matiašovic J.¹, Volf J.¹, Rychlík I.¹, Faldyna M.^1,2

¹Veterinary Research Institute, Brno, Czech Republic; e-mail: zelnickova@vri.cz

²University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic

The first aim of this work was to introduce the seven-color flow cytometry for evaluation of mononuclear phagocyte (MP) subpopulations in pigs. Moreover, using this method, the changes in these subpopulations after in vivo salmonella infection were assessed in the bone marrow (BM) and peripheral blood (PB).

The antibodies against cell surface markers CD14, CD163, CD172α, CD203α, SWC8 and MHCII together with viability staining with 7-aminoactinomycin D were used for the identification of MP subpopulations. The samples were analyzed by seven-color flow cytometer FACS Aria.

Since three of above mentioned primary antibodies shared the same subisotype and they were unfortunately not available as direct conjugates with appropriate fluorochromes the new technique for antibody labeling (the Zenon technology) had to be introduced and successfully used in pig.

Initially, all MP were identified as CD172α⁺ SWC8^- CD203α^- viable mononuclear leukocytes. After that the MP subpopulations were discriminated by CD14, CD163 and MHCII markers. Four distinct MP subpopulations indicating three completely distinct maturation pathways were found in the BM. The (CD172α^lo)CD14^- CD163^- MHCII^- BM MP continuously developed into (CD172α^lo) CD14^- CD163^- MHCII⁺ or (CD172α^hi) CD14⁺ CD163^- MHCII^- or (CD172α^hi) CD14⁺ CD163⁺ MHCII^- cells. Since three corresponding MP subpopulations were then identified in the peripheral blood: (CD172α^lo) CD14^- CD163^- MHCII⁺ dendritic cells and (CD172α^hi) CD14⁺ CD163^- MHCII^+/- or (CD172α^hi) CD14⁺ CD163^- MHCII^+/- monocytes, we can suggest that all these three PB MP subpopulations develop by unique pathways in the BM.

In Salmonella infected piglets, the only population of (CD172α^hi) CD14⁺ CD163^- MHCII^- cells was changed, indicating the role of this population in pathogenesis of salmonella infection in pigs.

This work was supported by grant GA524/08/1606.

Flow cytometric analysis of TNF-related apoptosis inducing ligand (TRAIL) internalization

Skender B.^1,2, Vondálová Blanářová O.^1,2, Jelínková I.^1,2, Hofmanová J.^1,2, Souček K.^1,2, Kozubík A.^1,2

¹Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic; e-mail: belma@ibp.cz 

²Faculty of Science, Masaryk University, Brno, Czech Republic

TNF-related apoptosis inducing ligand (TRAIL) is a transmembrane protein belonging to the Tumor Necrosis Factor (TNF) superfamily. Its expression is characteristic especially for various cells of immune system. TRAIL transduces an apoptotic signal by binding to its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2), which both contain a death domain as a part of their cytoplasmatic tail enabling them to bind death-inducing signaling complex (DISC), which leads to the activation of procaspase-8. Two complementary pathways were shown to lead from caspase-8 activation to execution of apoptosis (Lacour S. et al, 2003). One of them is independent on mitochondria (extrinsic pathway) and the second includes amplification of the apoptotic signal on the mitochondrial level (intrinsic pathway).

TRAIL induces apoptosis in cancer cells while sparing most of normal cells. Thus, in contrast to other members of the TNF superfamily, TRAIL administration in vivo is relatively safe. The absence of toxic side effects in addition to its anti-tumor properties makes this protein promising agent in anti-cancer therapy (Ishibashi M. et Ohtsuki T., 2008).

Recent experiments showed that TRAIL-induced apoptotic pathway can be also activated by internalization of this ligand with adaptor molecule DISC (Kohlhaas S. L. et al, 2007). These findings brought a new insight in TRAIL signaling. In this work, we evaluated method for measurement of internalized TRAIL using flow cytometry in two different cancer cell lines - PC3 (prostate cancer cell line) and HCT-116 (colon cancer cell line). We used Alexa Fluor 647 Microscale Protein Labeling Kit for TRAIL labeling. Sucrose was used as a negative control upon its ability to completely block receptor internalization while inducing minimum levels of apoptosis. Using fluorescently-labeled TRAIL we were able to quantify internalization of the ligand which can help us to better understand this new signaling pathway.

This work was supported by grants No. 1QS500040507 IGA ASCR and 301/07/1557, 524/07/1178 GACR.

References

1. Ishibashi M, Ohtsuki T. Studies on Search for Bioactive Natural Products Targeting TRAIL Signaling Leading to Tumor Cell Apoptosis. Medicinal Research Reviews 2008; 28: 688–714.

2. Kohlhaas SL, Craxton A, Sun X-M, Pinkoski MJ, Cohen GM. Receptor-mediated Endocytosis Is Not Required for Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis. J Biol Chem 2007; 282: 12831–12841.

3. Lacour S, Micheau O, Hammann A, Drouineaud V, Tschopp J, Solary E, Dimanche-Boitrel M-T. Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells. Oncogene 2003; 22: 1807–1816.

Time-lapse living cells microscopy of cell line K562 for study of CML-associated proteins dynamics – immobilization of suspension cultured cells

Potěšilová M., Vařecha M., Stejskal S., Koutná I., Kozubek M.

Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Brno, Czech Republic; e-mail: m.potesilova@gmail.com

Chronic myelogenous leukemia (CML) is characterized by genetic abnormality, the Philadelphia chromosome carrying BCR/ABL gene. This gene generates an aberrant kinase p210 (or less frequent type p190 or p230) whose unregulated activity mainly participates in the hematopoietic stem cells malignant transformation (Barnes and Melo, 2002). To fully understand the leukemogenesis it is important to describe not only p210 kinase function, but also the roles of native ABL1 and BCR kinases in the cell. We focus on localization and dynamics of these proteins in living leukemic cells that can bring new insights into the protein function.

Time-lapse microscopy of living cells is a method allowing us to study proteins in their natural environmental conditions in real time. Thanks to fluorescent proteins it is possible to examine protein localization and moreover protein dynamics or protein-protein interactions in living cells (Rustom et al., 2000). Adherent cells are mainly used for this type of methods, since these cells are not mobile or subject to gravity. However, we work with suspension cells such as K562 or CD34⁺ hematopoietic stem cells in our CML study.

Living-cell microscopy of non-adherent cells is complicated and seldom done, because it is necessary to immobilize suspension cultured cells to coverglass to protect them from the microscope stage movements, objective focus, and media flow caused by additional pipetting. It is also crucial to maintain the viability of the cells during such experiment and not to alter it by immobilization techniques. There are different ways to immobilize cells in 2D or 3D mode. For 2D mode it is possible to use surfaces coated with poly-L-lysine, chemically etched glass, fibronectin, collagen, and so forth. Unfortunately these surfaces also change shape and viability of suspension cells. Thus they are not very suitable for living-cells experiments. Therefore, we tested the immobilized culture of K562 cells on poly-L-lysin coated LabTec chamber. We compared results with novel method using oleyl poly (ethylene glycol) ether (SUNBRIGHT OE-020CS) modified LabTec chamber, suitable for studying suspension cells with maximal viability, unaffected cell growth, and maintained cell shape (Kato et al., 2003).

This work was supported by the Ministry of Education of the Czech Republic grants NPVII (2B06052) and LC535. 

References

1. Barnes DJ, Melo JV. Cytogenetic and molecular genetic aspects of chronic myeloid leukaemia. Acta Haematol 2002; 108: 180–202.

2. Kato K, Umezawa K, Funeriu DP, Miyake M, Miyake J, Nagamune T. Immobilized culture of nonadherent cells on an oleyl poly(ethylene glycol) ether-modified surface. Biotechniques 2003; 35: 1014–1018, 1020–1021.

3. Rustom A, Gerlich D, Rudolf R, Heinemann C, Eils R, Gerdes HH. Analysis of fast dynamic processes in living cells: high-resolution and high-speed dual-color imaging combined with automated image analysis. Biotechniques 2000; 28: 722–728, 730.

Detection of cellular prion protein on surface of lymphocytes and circulating dendritic cells is affected by blood storage

Glierová H, Holada K.

Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic; e-mail: hana.glierova@lf1.cuni.cz

Prion diseases, also called Transmissible Spongiphorm Encephalopathies (TSE), are fatal neurodegenerative disorders affecting humans and animals. The basic event in pathogenesis of prion infection is conversion of normal cellular prion protein (PrPc) into its pathological form, infectious prion protein (PrPtse). PrPc is expressed on various cell types including blood cells however little is known about the nature and behavior of prions in blood. The most common human TSE is Creutzfeldt-Jakob disease (CJD) occurring with frequency 1–2 cases/million inhabitants per year. New variant form of CJD (vCJD) was transmitted to humans by food infected with BSE. Four cases of secondary vCJD transmitted by blood transfusion highlight the need for development of screening test for prions and raise concerns about the safety of blood products.

We assume that prion infection may lead to expression of PrPtse on a distinct sub-population of blood cells to the level detectable by flow cytometry using conformation sensitive antibodies. Due to scarcity of CJD patients the studies of PrPtse/PrPc expression may involve shipment of blood samples from several collaborating centers. Our study was aimed at definition of the best conditions for shipment of blood and on examining the effect of different conditions on the level of nonspecific binding of PrPtse specific antibody V5B2.

The effect of anticoagulant and storage temperature on the binding of anti-prion antibodies to selected populations of blood leukocytes was studied. Blood of ten healthy donors was collected into standard EDTA and citrate vacutainer tubes. The blood was split into three parts. First was analyzed within 4 hours of blood collection, the other two were stored overnight at different temperatures (RT, 4 °C) before analysis. The analysis of PrP expression was accomplished by quantitative three color flow cytometry utilizing R-phycoerythrin conjugated monoclonal antibodies AG4 (PrP 31–51), AH6 (PrP 159–175) and V5B2 (PrP 214–225). Two antibody panels were used: first for identification of circulating dendritic cells which were defined as lineage markers negative (CD3, CD14, CD16, CD19, CD20, CD56, all FITC) and HLA-DR (PE DY647) positive cells, second antibody panel utilized CD3 (PerCP), CD19 (APC) and CD56 (FITC) to define T-cell, B-cell and NK-cell populations, respectively.

Significant effect of studied factors on the detection of surface PrPc was found. Generally, the storage of blood led to decrease in PrPc expression, which was more pronounced at RT and with AG4 detection. The extent of decrease was cell population specific ranging from almost complete loss to negligible alteration. Differences in nonspecific binding of V5B2 were not detected.

Both, the choice of anticoagulant and storage temperature influence the detection of PrPc on sub-populations of blood cells. If shipment of blood samples cannot be avoided, than anticoagulation with EDTA and temperature 4 °C are conditions of choice. Changes introduced by storage of blood did not introduce artificial binding of V5B2 suggesting the significance of its eventual binding to cells in CJD blood.

This work was supported partly by grants of Czech Science Foundation 310/05/H533 and 310/08/0878.

Investigation of bacterial populations in freshwater outdoor aerated and non-aerated microcosms

Mikula P.¹, Maršálek B.^1,2

¹Department of Experimental Phycology and Ecotoxicology, Institute of Botany ASCR, Brno, Czech Republic; e-mail: premysl.mikula@centrum.cz

²Research Centre for Environmental Chemistry and Ecotoxicology (RECETOX), Masaryk University, Brno, Czech Republic

The aim of our study was to investigate total bacterial counts and characteristics of bacterial populations in freshwater outdoor microcosms with different aeration regimes. Natural sediment from Nove Mlyny reservoir was added into twelve outdoor microcosms (about 80 litres of volume). Microcosms were subsequently filled with natural water containing plankton communities. Six microcosms were continuously aerated by an air pump (group 1), whereas remaining non-aerated microcosms were covered to provide minimal oxygen concentrations (group 2). After aeration procedure water from all microcosms was sampled and total bacterial counts as well as the ratio between HNA (high nucleic acid content) and LNA (low nucleic acid content) bacteria were investigated by means of flow cytometry (Gasol et al., 1999). For the measurements CyFlow ML cytometer (Partec GmbH, Muenster, Germany) equipped with blue laser emitting at 488 nm (20 mW) was used. Samples were stained with SYBR Green I using slightly modificated Marie’s protocol (Marie et al., 1997). Bacteria were enumerated according their side scatter (SSC) and green fluorescence (FL1, 527/30 nm). Red fluorescence (FL2 630 LP) was also measured for the discrimination of phytoplankton cells. Total bacterial counts in individual microcosms differed greatly not only between both groups tested, but also within each group (4.01 × 10⁵ to 3.15 × 10⁶). On the other hand, interesting data were obtained when we studied HNA/LNA ratio. Whereas majority of HNA bacteria was detected in all microcosms from group 1 (HNA/LNA ratio 2.62), in group 2 microcosms which weren‘t aerated, this trend was not apparent (HNA/LNA ratio 0.98). Our results confirmed the hypothesis that the aeration of water column can strongly influence bacterial populations.

This work was supported by Institute of Botany ASCR (Project No. AV0Z60050516) and the Ministry of Education, Youth, and Sports of the Czech Republic (Project No. IM6798593901).

References

1. Gasol JM, Zweifel UL, Peters F, Fuhrman JA, Hagstrom A. Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria. Appl Environ Microbiol 1999; 65: 4475–4485.

2. Marie D, Partensky F, Jacquet S, Vaulot D. Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Appl Environ Microbiol 1997; 63: 186–193.

Expansion of adoptively transferred activated naive CD8⁺ T cells by IL-2/anti-IL-2 mAb immunocomplexes as a scale of immunosupressive activity of anti-cancer drugs

Tomala J.¹, Chmelová H.¹, Strohalm J.², Etrych T.², Říhová B.¹, Kovář M.¹

¹Laboratory of Tumor Immunology, Department of Immunology and Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; e-mail: tomala@biomed.cas.cz

²Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic

Recently devised method for selective expansion of various populations (Boyman et al, 2006), based on the finding that the biological activity of IL-2 in vivo could be greatly enhanced by association with certain anti–IL-2 mAbs, opens broad new possibilities of application in different fields, ranging from vaccination and transplantation to treatment of autoimmune diseases and tumours.

For two out of three anti-IL-2 mAbs avaible, injecting IL 2–mAb complexes led to marked and selective proliferation of MP CD8⁺ cells and NK cells, i.e, cells expressing low-affinity IL-2Rs.

Thus, we explored the possibility to use IL-2 immunocomplex-driven expansion of CD8⁺ T cells to measure immunosupressive activity of anti-cancer drugs.

We have used purified CFSE labeled CD8⁺ OT-1 cells for adoptive transfer into B6/SLJ congenic mice with appropriate stimulation with peptide (SIINFEKL) and treatment with S4B6/IL-2 immunocomplexes. Subsequently, free doxorubicin a various polymer-bound doxorubicin conjugates have been applied. We have measured clonal expansion and expression of selected surface markers.

We have shown that the expansion of OT-I CD8⁺ T cells driven by S4B6/IL2 immunocomplexes could be utilized as an appropriate method in determining immunosuppressive effect of anti-cancer therapeutics. As an example, we demonstrated that free doxorubicin has strikingly strong immunosuppressive impact on expanding CD8⁺ T cell population, in comparison to polymer bound doxorubicin conjugates, which inhibit expansion of CD8⁺ T cells much less.

References

1. Boyman O, Kovar M, Rubinstein MP, Surh CD, Sprent J. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 2006; 311: 1924–1927.

NK and NKT cells increase after rituximab treatment in patients with rheumatoid arthritis

Kryštůfková O.¹, Król P.², Hulejová H.¹, Pavelka K.¹, Šenolt L.¹, Vencovský J.¹

¹Laboratory of Flow cytometry and Clinical Department, Institute of Rheumatology and Dept. of Rheumatology 1^st Medical Faculty, Charles University, Prague; e-mail: krys@revma.cz

²Department of Pediatrics and Adolescent Medicine, General Teaching Hospital, Charles University, 1st Medical School, Prague, Czech Republic

Rituximab (RTX) is a chimeric monoclonal antibody registered for treatment of rheumatoid arthritis (RA) which induces rapid depletion of B cells expressing CD20. The killing of B lymphocytes is mediated by NK cells via antibody-dependent cell-mediated cytotoxicity. The initial consumption of NK cells is followed by their enhanced production and extended life-span. Lower numbers of NK cells in peripheral blood of patients with active RA and their expansion in inflamed synovium were reported. NKT cells, a heterogeneous T cell population characterized by co-expression of NK and T cell markers, are involved in regulation of immune response by rapid secretion of cytokines. Reduced levels of NKT cells have been detected in some Th1 mediated autoimmune diseases, such as RA. Increase of relative and absolute values of circulating NK and NKT cells during RTX treatment was reported in SLE patients. The aim of the study was to evaluate changes in peripheral blood NK and NKT cells during RTX treatment of RA patients and relate these to clinical responses.

Twenty-five RA patients were treated with two infusions of 1 g RTX 2 weeks apart and followed up at two month intervals. Profile of PBMC (obtained by gradient centrifugation) was measured by multicolor flow cytometry at baseline, after 8 weeks and 24 weeks of RTX application. 10⁶ cells were stained by 7- colors panel and acquired on CyAn ADP. After the sequential gate for singlets, lymphocytes and CD45⁺CD14^- cells, the percentage from lymphogate was analyzed. B cells were defined as CD19⁺, NK cells as CD3^-CD16/56⁺,NKT as CD3⁺CD16/56⁺ and CD3^bright cells were considered to be γδ T-cells.

All patients reached B-cell depletion (from baseline 0.14 × 10⁹/l to 0.002 × 10⁹/l at 8 week) after baseline. Eighteen patients showed a good or moderate EULAR response at week 16. Twenty patients were re-treated with the median 8 months from baseline.

At week 8, a significant increase of NK and NKT frequency was observed – for NK from baseline 10.6% to 14.5% (p = 0.001) and for NKT from baseline 5.7% to 7.0% (p = 0.006). Increase in CD3 ^bright – γδT cells (from 1.2% to 2%; p = 0.025) was also recorded. Increase in absolute numbers of NK cells from 0.17 to 0.23 × 10⁹/l (p = 0.011) and of CD3 ^bright cells from 0.02 to 0.03 (p = 0.014) was seen at week 8. At week 24, no additional significant changes of all the followed lymphocyte subsets were found. For evaluation of the effect of NK and NKT cell elevation at week 8 (expressed as percentage of baseline values) on the clinical response, they were compared with the difference of DAS28 score from baseline to week 16. Interestingly, change in DAS28 score correlated negatively with the change of NK cell percentage (rs = -0.42; p = 0.038) and positively with the change of absolute NKT cell numbers (rs = 0.44; p = 0.006). The latter was probably result of the changes of absolute lymphocyte numbers, which correlated strongly with clinical response (rs= 0.63; p = 0.0002).

Conclusion:NK and NKT cells increase after B cell depleting therapy in RA. The negative correlation of the change of NK cells with the change in DAS28 score suggests an ongoing consumption of NK cells in week 8 followed with better clinical response in week 16. Since the increase in NKT cells correlates well with improvement in disease activity, a regulatory role of NKT cell could be important in mediating the effect of anti-CD20 treatment or reflect the decrease of disease activity in the sites of inflammation. The significance of an increase in γδT cell numbers needs further clarification.

Institutional support VZ MZ 0002372801 Ministry of Health, CR.

Different properties of cord blood cells of newborns of healthy and allergic mothers

Hrdý J.¹, Novotná O.¹, Kocourková I.², Šterzl I.¹, Prokešová L.¹

¹Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic; e-mail: jirka81@seznam.cz

²Institute for the Care of Mother and Child, Prague, Czech Republic

Allergic diseases belong to common illnesses with still increasing incidence over recent decades which does not seem to stop this trend. Allergic diseases represent serious medicinal, social and economical problem. The need for the development of prevention strategies is of particular interest. A clear understanding of the immunological processes promoting Th2-polarized atopic sensitization in early life is of essential importance. It is also necessary to keep in mind the strong genetic background of allergic diseases. In our study, we tried to identify some early prognostics markers in cord blood cells pointing to increased risk of future allergy development which could serve to early introduction of preventive measures.

Properties of cord blood cells of children with high risk of allergy (children of allergic mothers) were compared with cord blood cells of children of healthy mothers. We tested in vitro proliferation of cord blood mononuclear leukocytes (CBML) by ³H thymidine incorporation. Expression of cytokines (IL-1beta, IL-2, IL-4, IL-8, IL-10, IL-13, IFN-gamma, TNF-alpha, TGF-beta and EGF) in nonstimulated and polyclonally stimulated CBML was measured by real-time PCR. Concentration of cytokines mentioned above was tested by ELISA in cord blood sera of children of healthy and allergic mothers and in culture supernatants of CBML stimulated by Bacillus firmus (a gram-positive bacterium) or PHA. Cord blood myeloid dendritic cells – mDCs (grown in vitro from CD14 positive monocytes) were characterized by gene expression of activation markers (CD80, CD83, CD86), IDO (indoleamine 2, 3-dioxygenase), TNF-alpha, IL-6 and cytokines of IL-12 family. Proportion of Tregs in cord blood of children of healthy and allergic mothers was compared by flow cytometry. T-test was used for statistical analyzing normally distributed data otherwise non-parametric Mann-Whitney test was exploited.

CBML of children of allergic mothers have significantly higher both spontaneous and polyclonally stimulated proliferation activity pointing to possible increased promptness of future allergy onset (1). Nonstimulated cord blood cells of allergic group are characterized by increased expression of IL-10, IL-13, EGF and decreased expression of IL-1beta, IL-2, IL-4, IL-8, IFN-gamma, TNF-alpha and TGF-beta in comparison with CBML of children of healthy mothers. Expression of cytokines after in vitro stimulation of CBML is generally increased in allergic group in comparison with healthy one. These two groups differ also in serum cytokine levels although only IL-10 and EGF were significantly different: IL-10 was increased and EGF decreased in cord sera of allergic group. Increased proportion of mDC expressing CD83 after LPS stimulation was found in allergic group. Tendency to increased IL-6 expression after LPS stimulation was found in mDC of children of allergic mothers. We did not observe significant difference in proportion of Tregs between cord bloods of children of healthy and allergic mothers.

Allergic phenotype was apparent already on the level of cord blood cells. Higher lymphocyte proliferation activity and higher stimulation readiness of mDC and lymphocytes of cord blood of children of allergic mothers imply easier allergy induction. Also, significantly decreased levels of EGF in newborns of allergic mothers could negatively influence maturation and permeability of mucosal membranes of these children and support thus allergen penetration.

This work was supported by grants of Ministry of Education, Youth and Sports of the Czech Republic MSM0021620806 and Grant Agency of the Czech Republic 310/08/H077.

References

1. Zizka J, Hrdy J, Lodinova-Zadnikova R, Kocourkova I, Novotna O, Sterzl I, Prokesova L. Effect of breast milk of healthy and allergic mothers on in vitro stimulation of cord blood lymphocytes. Pediatr Allergy Immunol 2007; 18: 486–494.

Modulation of cytokinetic parameters and ECM-mediated signaling in colon adenocarcinoma cells, HT-29, in dependence on the type of cell culture surface

Kočí L.¹, Procházková J.^1,2, Hofmanová J.¹, Hýžďalová M.¹, Kozubík A.¹

¹Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i, Brno, Czech Republic; e-mail: koci@ibp.cz

²Veterinary Research Institute, v.v.i, Brno, Czech Republic

Since colorectal cancer represents one of the most common types of tumor disease in the Western world, the development of various therapeutical approaches is therefore the necessity of crucial importance. Colon carcinogenesis is a multistep process, which requires deregulation of essential cellular mechanisms including e.g. cell proliferation, cell cycle control and communication between cells and components of extracellular matrix (ECM). In this study, we have analyzed aforementioned cytokinetic parameters in an in vitro model of colorectal adenocarcinoma represented by HT-29 cells, in dependence on the type of cell culture surface.

To assess the importance of cell anchorage type, we have cultivated cells in ECM protein-coated (collagen IV and fibronectin) and non-coated (control) tissue culture plastic. First, the adhesion rate of HT-29 cells has been tested with xCELLigence system (Roche Diagnostics) using different culture surfaces. A special attention has been paid to determination of time interval, which would be sufficient for analysis of cell adhesion and for description of early events (up to 6 h) associated with formation of cell-ECM contacts. During this time interval, we observed apparently enhanced adhesion of HT-29 cells only on the cell culture surface coated with collagen, but not with fibronectin. Second, these experiments help us to establish approximate doubling time of HT-29 cells (around 8 hours). Third, our results further indicate that the type of cell culture surface has no significant effect on cell number or cell cycle distribution, while adhesion rate of HT-29 cells seems to be strongly enhanced, especially in the presence of collagen IV. Finally, expression of some molecules implicated in the cell-ECM signaling has been tested at different levels (mRNA, protein).

Taken together, our data imply that HT-29 cells could be used as an appropriate in vitro model for studying the role of cell-ECM contacts in the context of colon carcinogenesis.

This work was supported by grants 305/09/1526 GACR, 524/07/1178 GACR and 1QS500040507 IGA AV ČR.

Telomerase (pTERT) mRNA expression in the pig granulosa cells in vitro

Tománek M.¹, Kott T.², Chronovska E.¹, Kottová E.²

¹Institute of Animal Science, Department of Biology of Reproduction, Prague 10 - Uhříněves, Czech Republic;e-mail: tomanek.milan@vuzv.cz 

²Institute of Animal Science, Department of Molecular Genetics, Prague 10 – Uhříněves, Czech Republic

Telomerase plays an essential role in cell viability and has been defined as a key factor for the regulation of cell proliferation and senescence. Recent studies showed that under various physiological and pathological conditions the dynamics of telomerase expression and activity in proliferating, regenerating or differentiating tissue is regulated by cellular microenvironment and growth factors (Bayne and Liu, 2005). We described recently telomerase enzymatic activity in pig granulosa cells in vitro and we suggested that its activity and correlation with cell proliferation and differentiation may be differentially regulated in small and large developing ovarian follicles (Tomanek et al, 2008). The aim of presented work was to develop a new RT-PCR method of pig telomerase (pTERT) mRNA quantification and its use for the study of pTERT mRNA expression in the pig follicular granulosa cells in vitro.

Granulosa cells (GC) were isolated from small (SF-GC) and large (LF-GC) follicles and cultured for 96 h in vitro in DMEM/F12 medium supplemented with 2% FBS, ITS, gentamycin, testosterone (10^-8M), EGF (10 ng/ml) and pFSH (50 ng/ml). Telomerase activity was assayed with the modified protocol based on the TRAPEZE^® Telomerase Detection Kit, (Chemicon). For RT-PCR, the total RNA was isolated from granulosa cells with the use of Total RNA Chemistry method and 6100 Nucleic Acid PrepStation (Applied Biosystems). Primers and TagMan MGB probes for pTERT were designed with the Primer Expres ver. 3 Program. The mRNA was reverse transcribed into cDNA with the High Capacity cDNA Archive Kit and RT-PCR was performed on the ABI 7500 Fast Real-Time PCR System (Applied Biosystems).

Freshly isolated SF-GC expressed higher levels of pTERT mRNA than LF-GC (3.363 vs. 1.834 RU) as well as higher levels of telomerase activity (112.11 vs. 71.20 TPG/prot.). During in vitro culture, pTERT mRNA levels in SF-GC increased in control and FSH treated cells (5.815 and 6.405 RU) and slightly decreased in EGF treated cells (2.723 RU). On the other hand, LF-GC expressed significantly lower pTERT mRNA levels (1.112, 0.78 and 0.95 for control, EGF and FSH) as well as the levels of telomerase activity (8.32, 9.66 and 11.12 TPG/mg prot. for control, EGF and FSH, respectively). In conclusion, we suggest that telomerase expression and activity in pig follicular granulosa cells is related to their proliferative potential and function in developing ovarian follicles and pTERT mRNA expression and telomerase activity decline in highly differentiated, luteinized granulosa cells.

Supported by GACR 523/05/2062 and MZE 0002701401 grants.

References

1. Bayne S, Liu JP. Hormones and growth factors regulate telomerase activity in ageing and cancer. Mol Cell Endocrinol 2005; 240: 11–22.

2. Tománek M, Chronowska E, Kott T, Czerneková V. Telomerase activity in pig granulosa cells proliferating and differentiating in vitro. Anim Reprod Sci 2008; 104: 284–298.

Interaction of quaternary benzo[c]phenanthridine alkaloids with DNA

Hammerová J.¹, Táborský P.², Urbanová J.², Gregorová J.³, Táborská E.³, Slaninová I.¹

¹Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; e-mail: ipokorna@med.muni.cz

²Department. of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic

³Department of Biochemistry, Faculty of Medicine, Masaryk University, Brno, Czech Republic

Alkaloids are low-molecular weight nitrogen containing secondary plant metabolites, usually with heterocyclic structure. QBAs are group of alkaloids occurring in some species of Fumariaceae, Papaveraceae, Ranunculaceae and Rutaceae families. Extracts from some plants containing QBAs are used in folk medicine as antimicrobial, antifungal and anti-inflammatory agents (for review see Šimánek, 1985). In addition to wide range of biological activities, QBAs also display interesting colour properties (from yellow to dark red) and fluorescence properties (Kovář et al., 1985; Slaninová et al., 2008). The chromophores responsible for fluorescence are highly condensed aromatic rings with electron-donor substituents containing oxygen. The other important property of these alkaloids is the ability to interact with DNA.

The interaction of SA with DNA has been described by several authors during the last decade. Although the concept of intercalation is generally accepted, also other types of interactions have been considered, eg. out side binding of SA to the phosphate backbone of the double helical DNA or its binding to G-C rich regions of DNA (for review see Slaninová et al., 2008).

Recently we have described that macarpine (MA), chelirubine (CHR), sanguirubine (SR) and sanguinarine (SA) stain nucleic acids in living cells (Slaninová et al., 2007). We concluded that they can be used as supravital fluorescent DNA probes both in fluorescence microscopy and flow cytometry including multiparameter analysis of peripheral blood and bone marrow. The best DNA staining properties showed MA. MA binds DNA stoichiometrically and can report the DNA content. QBA are excitable using common argon lasers (488 nm) emitting at a range of 575–755 nm (i.e. fluorescence detectors FL2-5).

In order to characterize QBA–DNA interactions, we have carried out in vitro studies of binding of QBA to double stranded DNA (Urbanová et al., 2009) using fluorescence and UV-Vis spectroscopy. We have studied the spectral properties of SA, chelerythrine (CHE), CHR, SR, chelilutine (CHL), sanguilutine (SL) and MA in presence and in absence of calf thymus DNA. Generally, QBAs have at least four absorption maxims in UV-Vis spectrum between 250 and 550 nm. First maximum is below 300 nm, second dominant peak is usually between 310 and 350 nm, the third absorption band is about 400 nm and merges to a wide band absorption between 450 and 520 nm (the last two bands are responsible for the colors of the alkaloids). Two different emission bands were found in the fluorescence spectra of the studied QBAs after excitation by light with a wavelength of about 330 nm (second excitation band). First, the high energetic band is situated between 400 and 500 nm, second, the low energetic band, is present between 500 and 700 nm. Intensities of these bands changes at the presence of DNA and these changes are characteristic for each alkaloid. Results from these experiments confirmed a great affinity of the studied alkaloids to DNA – association constants (logK₁₁) are about 5. Although calculated association constants for all alkaloid-DNA systems were comparable, we observed dramatic differences in fluorescence emission of all studied alkaloids in presence of calf thymus DNA in comparison to fluorescence of free alkaloids. The most remarkable were changes in emission spectra of MA, CHRandSR(Urbanová et al., 2009).

The results from all our studies, fluorescence spectroscopy, fluorescence microscopy and flow cytometry, confirmed great affinity of QBAs to double stranded DNA that is accompanied by changes in fluorescence properties. These characteristics of QBA provide possibilities to their use in analytical and bioanalytical chemistry and in fluorescence microscopy and flow cytometry.

This work was supported by the Czech Science Foundation (project No. 525/08/0819) and the Ministry of Education of the Czech Republic (VZ MSM0021622415 and LC06077).

References

1. Kovář J, Šimek K, Kožoušková E, Klukanová H. Fluorescence properties of some isoquinoline alkaloids. Collect Czech Chem Commun 1985; 50: 1312–1328.

2. Šimánek V. Benzophenanthridine alkaloids. In: Brossi, A. (ed.) The Alkaloids 26. Orlando: Academic Press 1985; 185–240.

3. Slaninová, I, Slanina, J, Táborská, E. Fluorescenční vlastnosti kvartérních benzo[c]fenanthridinových alkaloidů a jejich využití jako supravitálních DNA sond. Chem. Listy 2008; 102: 427–433.

4. Slaninová I, Slanina J, Táborská E. Quaternary Benzo(c)--phenanthridine alkaloids – novel cell permeant and red fluorescing DNA probes. Cytometry Part A 2007; 71A: 700–708.

5. Urbanová J, Lubal P, Slaninová I, Táborská E, Táborský P. Fluorescence properties of selected benzo[c]phenantridine alkaloids and studies of their interaction with CT DNA. Analytical and Bioanalytical Chemistry 2009; 997–1002.

Evaluation of Ca²⁺ signaling in hepatocytes by flow cytometry

Uhrová D, Kostecká P, Krejčová V, Zídek Z, Kmoníčková E.

Institute of Experimental Medicine, Academy of Science of Czech Republic; e-mail: jollydan@post.cz

Flow cytometry has previously been used to measure calcium flux in other cell types, including eosinophils, lymphocytes etc. Here we describe that calcium flux can be detected in rat hepatocytes. For modulation of calcium signaling, SERCA (sarco/endoplasmic reticulum Ca²⁺ ATPase) inhibitor, thapsigargin, was used in various concentrations (0.1; 0.4; 1.0 μM) and cells were incubated in medium with 1 mM Ca²⁺ or in Ca-free medium. Hepatocytes were incubated in the presence of calcium indicator dye Fluo-4/AM, added from 1 mM stock solution in anhydrous dimethyl sulfoxide (DMSO). Suspension of cells (10⁶/ml) were loaded with 1 μM Fluo-4/AM (Molecular Probes, Invitrogen) for 45 min in the dark at 37 °C. After incubation hepatocytes were washed twice at room temperature in assay buffer with 1mM Ca²⁺ or in Ca-free buffer to remove extracellular dye. Analyses were performed with the FACSAria flow cytometer (Becton Dickinson). Green fluorescence was measured over a period of 70 s. 

Our pilot measurements yield promising data for measurement of Ca²⁺ signal in hepatocytes by flow cytometry with Fluo-4/AM dye. 1. Low signal of fluorescence was recorded in the Ca-free samples. On the other hand, higher fluorescence intensity was found in hepatocytes incubated with 1 mM Ca²⁺ which reflects influx of extracellular calcium. 2. In last named experimental conditions the dependency of measured signal on concentration of SERCA inhibitor is apparent.

However, there are some complications during on-line monitoring: hepatocytes represent dishomogenous cell population, are fragile for manipulation and can aggregate. The major problem represents their size and quick sedimentation. Nevertheless we can assume that this method is suitable for measuring of Ca²⁺ signaling in hepatocytes and further experiments are in progress.

This work was supported by: GA ČR 304/07/1129; GA ČR 309/08/H079; AVOZ 50390 703.

P14

Aberrant CEACAM6 influences integrin-ligand interactions and survival of leukemic cells

Kanderová V, Hrušák O, Kalina T.

CLIP-Cytometry, Charles University, 2^nd Medical School, Prague, Czech Republic; e-mail: kanderov@seznam.cz

Signal exchange between cell and its microenvironment is essential for proliferation, differentiation, survival, apoptosis or migration. Adhesion molecules of CEA (carcinoembryonic antigen) family initiate signaling pathways, which influence all above mentioned processes in malignant GI tissues and in nonmalignant granulocytes. GPI-anchored CEACAM6 is expressed on the tumors of colon, stomach, pancreas, lungs and breast, and the expression is often associated with a poor clinical outcome (Hammarstrom, 1999). Within the framework of the so-called membrane lipid rafts, CEACAM6 co-localizes with beta1 integrin and activates the anti-apoptotic signaling pathways (PI3K/AKT) in the colon and pancreatic carcinomas (Chan and Stanners, 2007). Antibodies targeting the N domain of CEACAM6 inhibit the adhesion and metastasis in the in vitro/in vivo models of all the above listed tumors (Blumenthal, 2005). Within the hematopoietic system CEACAM6 is expressed on the granulocytes and is involved in the homotypic and heterotypic adhesion and Ca²⁺ mediated signaling. It increases the adhesiveness of the granulocytes to the ECM components through the beta1 and beta2 integrin activation (Nair and Zingde, 2001). In addition, CEACAM6 is the most frequent aberrant myeloid marker in childhood B-precursor acute lymphoblastic leukemia (BCP ALL). In BCP-ALL CEACAM6 positively correlates with the chromosomal translocation BCR-ABL and with the hyperdiploidy. Based on the stable expression of CEACAM6 from the diagnosis to the relapse, a feature exploited for the detection of the minimal residual disease, it is possible to estimate its contribution to the chemoresistance and recurrence of the disease (Kalina, 2005).

In our study, we examined the underlying molecular mechanism of CEACAM6 signaling and its effect to leukemic cells behavior. We used a model of antibody-mediated clustering of CEACAM6 molecules and cocultivation of leukemic blasts on a stromal feeder layer, which in vitro approximates bone marrow microenvironment. We demonstrated that CEACAM6 clustering mildly enhanced surface expression of beta1 integrins (CD49e/CD29) and beta2 integrins (CD11a/CD18, CD11b/CD18, CD11c/CD18) and also increased the binding activity of beta1 and beta2 integrins to VCAM-1 and ICAM-1 ligands. Next, we detected enhanced phosphorylation of anti-apoptotic kinases p44/42 MAPK, Akt and of kinase p38 MAPK with a resembling dynamics. Clustering of CEACAM6 triggered apoptosis of ALL blasts. When ALL blasts were cocultured on stroma and treated with imatinib (BCR/ABL inhibitor) CEACAM6 signaling enhanced survival of blast cells.

Thus, for the first time, we show that aberrant expression of myeloid CEACAM6 is functionally engaged in communication between ALL blasts and their microenvironment. CEACAM6 clustering enhanced integrin surface expression and integrin-ligand binding, activated downstream signaling molecules p44/42 MAPK, Akt and p38 MAPK and influenced survival of ALL blasts. This can play at least a supportive role in malignant behavior of leukemic cells.

This work was supported by GA UK 7539/2007, VZ MŠMT 0021620813.

References

1. Hammarstrom S. The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol 1999; 9: 67–81.

2. Chan CH, Stanners CP. Recent advances in the tumour biology of the GPI-anchored carcinoembryonic antigen family members CEACAM5 and CEACAM6. Curr Oncol 2007; 14: 70–73.

3. Blumenthal RD, Hansen HJ. et al. Inhibition of adhesion, invasion, and metastasis by antibodies targeting CEACAM6 (NCA-90) and CEACAM5 (Carcinoembryonic Antigen). Cancer Res 2005; 65: 8809–8817.

4. Nair KS, Zingde SM. Adhesion of neutrophils to fibronectin: role of the cd66 antigens. Cell Immunol 2001; 208: 96–106.

5. Kalina T, Vaskova M, et al. Myeloid antigens in childhood lymphoblastic leukemia: clinical data point to regulation of CD66c distinct from other myeloid antigens. BMC Cancer 2005; 5: 38.

Differentiation and apoptotic response of colon cancer cell lines to short- and long-chain fatty acids

Stixová L,^1,2, Hofmanová J,^1,2, Netíková J,¹, Kozubík A.^1,2

¹Department of Cytokinetics, Institute of Biophysics, Acad. Sci. Czech Republic, v.v.i, Brno; e-mail: lenka@ibp.cz

²Department of Animal Physiology and Immunology, Institute of Experimental Biology, Fac. of Sciences, Masaryk University, Brno

Dietary lipids, especially omega-3 polyunsaturated fatty acids (PUFAs) and butyrate, the short-chain fatty acid produced by microbial fermentation of fibre, are important modulators of colon epithelial cell kinetics and play an important role in colon carcinogenesis. These compounds are thus considered as chemoprotective nutrients in colon cancer.

We compared the differentiation and apoptotic response of the colonic cell lines derived from human normal fetal tissue (FHC) and poorly differentiated adenocarcinoma (HCT-116) to sodium butyrate (NaBt), docosahexaenoic acid (DHA, 22 : 6, ω-3), and their combination. The effects of NaBt and DHA were different in these two cell lines. While in FHC cells NaBt induced G/G₁ arrest, differentiation and low level of apoptosis, in HCT-116 cells G₂/M arrest, no differentiation and high degree of apoptosis were detected. Moreover, in FHC cells significant potentiation of apoptosis (DAPI staining, fluorescence microscopy) accompanied by increased arrest in the cell cycle, cell detachment, and decrease of differentiation (detected by alkaline phosphatase activity and carcinoembryonic antigen expression) were detected after combined treatment with NaBt and DHA.

Apoptotic cells were also visualized by flow cytometry using theM30 antibody. M30/propidium iodide double staining revealed that FHC cells arrested in G/G₁ and HCT 116 cells arrested in G₂/M phases of the cell cycle enter apoptosis mainly from these respective phases of the cell cycle. Modulation of percent of cells with decreased mitochondrial membrane potential (flow cytometry, TMRE) corresponded only partially with apoptotic response. Detection of caspase-3 activity by both fluorimetry and flow cytometry suggests its role in both FHC cell differentiation and apoptosis.

Our results support the idea of the interaction of butyrate and PUFAs in the colon and suggest its diverse impact in normal or neoplastic epithelium with varied participation of extrinsic and intrinsic (mitochondrial) apoptotic pathways.

This work was supported by grants Nos. 524/07/1178 and 305/09/1526 GA CR, and 1QS500040507 IGA ASCR.

Multicolor detection of selected intra- and extra-cellular markers during neuroendocrine transdifferentiation of prostate cancer epithelial cells

Pernicová Z.^1,2, Lincová E.^1,2, Staršíchová A.^1,3, Kozubík A.^1,2, Souček K.¹

¹Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic; e-mail: pernicova@ibp.cz

²Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

³Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic

Prostate cancer is one of the world’s most extended types of cancer in men. At the beginning, patients with prostate cancer usually benefit from the conventional treatment with hormonal therapy, which embody in androgen ablation. But the disease recurs in more severe manner, the tumor is more aggressive and cancer cells acquire androgen independence. Each cell type presented in prostate epithelium is characterized by expression of specific intracellular and extracellular markers. One of the type of cells, present in both normal and cancer prostate epithelium, are neuroendocrine cells or neuroendocrine-like cells, respectively. These cells are able to secrete many biologically active factors and modulate cancer tissue microenvironment. Such modulations can probably contribute to acquisition of androgen-independence of prostate cancer cells.

In our study transdifferentiation of prostate cancer cell line LNCaP was induced by prolonged cultivation in androgen-depleted media. This led to induction of neuroendocrine-like phenotype of these cells. Using flow cytometry we showed that expression of neuroendocrine marker neuron-specific enolase was uniformly increased in whole population of cells, which undergo transdifferentiation. Interestingly, androgen depletion induced increase in expression of cytokeratin and vimentin, markers of epithelial and mesenchymal differentiation, respectively. While the expression of cytokeratin was uniformly increased in whole population, increased expression of vimentin was detected only in subpopulation of transdifferentiated cells. To prove functional induction of neuroendocrine transdifferentiation in LNCaP cell line during long term androgen ablation, we detected expression of serotonin and histamine, markers, which are secreted by neuroendocrine and neuroendocrine-like cells.

Further, we wanted to find out if expressions of selected extracellular molecules change as a result of transdifferentiation and if expressions of some of these extracellular molecules correlate with expression of vimentin. Using flow cytometry we detected expression of CD44 and CD133, which are known as markers of prostate stem cells and prostate cancer stem cells. Expression of each selected molecule had distinct pattern.

In conclusion, our results showed, that transdifferentiation of prostate cancer cells affects not only the expression of specific neuroendocrine markers, but also influences expression of other extracellular and intracellular molecules. This indicates that population of neuroendocrine-like LNCaP cells, which arise in consequence of transdifferentiation induced by androgen depletion in vitro, is heterogeneous and distinct subpopulations are presented.

This work was supported by MU Rector’s Program for Student’s Creative Activity Support, and grants Nos. 204/07/0834, 310/07/0961 of the Czech Science Foundation, grant No. NS9600-4 of the Ministry of Health of the Czech Republic, and grants Nos. AV0Z50040507 and AV0Z50040702 of the Grant Agency of Academy of Sciences of the Czech Republic.

MicroRNA in epithelial-mesenchymal transition of benign prostate hyperplasia cells

Lincová, E.^1,2, Pernicová, Z.^1,2, Staršíchová, A.^1,2, Kozubík, A.^1,2, Souček, K.¹

¹Department of Cytokinetics, Institute of Biophysics AS CR, Brno, Czech Republic; e-mail: lincova@ibp.cz

²Department of Experimental Biology, Masaryk University, Faculty of Science, Brno, Czech Republic

Epithelial–mesenchymal transition (EMT), in which epithelial cells lose their polarity and become motile mesenchymal cells, occurs during development and is viewed as an essential early step in tumor metastasis. Transcription factors and EMT regulators of the ZEB family are thought to be involved in tumor progression, thus having potential clinical interest. MicroRNAs (miRNAs) are an abundant class of small non-protein-coding regulators of gene expression that play an important role in tumorigenesis and, depending on their targets, can function as tumor suppressors or oncogenes. miR-200 family and miR-205, which regulate expression of ZEB transcription factors, have been found downregulated during EMT, suggesting an important role in inhibition of EMT. The aim of this study is to describe the role of miRNA in EMT of benign prostate hyperplasia cells.

EMT of BPH-1 cell line was induced by TGF-α1 treatment and assessed by cell morphology and expression of epithelial (E-cadherin) and mesenchymal markers (N–cadherin, vimentin) on both protein and mRNA level. Expression of ZEB1, ZEB2, miR-200 family and miR-205 was analyzed by qRT-PCR. The effect of miR-200 on ZEB proteins translation was assessed by luciferase assay.

Decrease in expression of microRNAs of miR-200 family and miR-205 was observed 48 hours after treatment, which correlates with expression of EMT markers. Significant changes in expression of ZEB1, ZEB2 and SNAI2 transcripts observed already after 24 hours of TGF-α1 treatment suggest a possible role of these transcription factors in early stages of the EMT process of nontumorigenic prostate epithelial cell line.

This work was supported by grants No. 204/07/0834, No. 310/07/0961 of the Czech Science Foundation and by the AS CR, grants no. AV0Z50040507 and AV0Z50040702 and by the Masaryk University Rector’s Program for Support of Students’ Creative Activity.

References

1. Gregory PA, Bert AG, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 2008; 10: 593–601.

Activation of caspases in cells lytically infected with vaccinia virus

Lišková, J, Mělková, Z.

Institute of Immunology and Microbiology, 1^st Medical Faculty, Charles University, Prague, Czech Republic; e-mail: zmelk@lf1.cuni.cz

Vaccinia virus is a member of poxvirus family consisting of large DNA viruses that replicate in cytoplasm of infected cells. Various strains of vaccinia virus have been used for vaccination against smallpox as well as for experimental vaccination with recombinant proteins expressed by this highly efficient and immunogenic vector. Despite of a wide use of vaccinia virus, the nature of vaccinia virus-induced cell death remains insufficiently characterized. In most cells types, vaccinia virus is considered to cause a lytic infection, an equivalent of necrosis, while in certain other cell types, it induces apoptosis. Activation of the apoptotic cascade characterized by proteolytic cleavage of pro-caspases into active caspases is a hallmark of apoptosis. However, we have observed caspase activation and/or activity during a lytic infection of epithelial cell lines BSC-40 and HeLa G with vaccinia virus (Kalbacova et al., 2008).

In this report, we explored the above described phenomenon in detail. The caspase activation was first characterized by flow cytometry using a fluorescent pan-caspase inhibitor FITC-VAD-FMK which binds irreversibly to the active site of activated caspases. The specificity of the assay was confirmed using a pretreatment of the cells with the non-fluorescent pan-caspase inhibitor Z-VAD-FMK, resulting in a decrease of fluorescent signal. In contrast, inhibitors of other proteases like calpain or cathepsins D and E remained without any effect in this assay, further demonstrating a specificity of the assay for caspases. Additionally, activation of calpain and cathepsins during infection with vaccinia virus was characterized using specific inhibitors and western blot analysis of the cleavage products. Consequently, specific inhibitors of individual caspases (inhibitors of caspase-1, 2, 3, 4, 6, 8, 9, 10, 13) were used in the above described flow cytometric assay to compete out the binding of the fluorescent inhibitor and to decrease the fluorescent signal. The fluorescence was highly decreased by pretreatment of the cells with specific inhibitors of caspases-2 and 4 – to a similar level like with Z-VAD-FMK, and somewhat decreased by pretreatment with the inhibitor of caspase-3. Inhibitors of other caspases remained without effect. The activity of caspases-2, 4 and 3 was then confirmed in lysates of infected cells using an in vitro cleavage assay involving specific fluorogenic substrates. Again, the specificity of each cleavage was confirmed using specific non-fluorescent inhibitors. Finally, the cleavage of caspase substrates, PARP and cytokeratin-18, was characterized in vaccinia virus-infected cells using a western blot analysis. While we found cleavage of cytokeratin-18, we did not observe any significant cleavage of PARP, a critical substrate of the executioner caspases. Thus, the lack of cleavage of PARP correlates with the final lytic outcome of vaccinia virus infection in these cells.

In conclusion, we describe activation and activity of caspases-2 and 4 in epithelial cells lytically infected with vaccinia virus. The role of these caspases during vaccinia virus infection will be discussed.

This work was supported by the Grant Agency of the Czech Republic – projects Nos. 310/05/0477 and 310/05/H533, and by the Ministry of Education of the Czech Republic – project No MSM0021620806.

References

1. Kalbacova M, Spisakova M, Liskova J, Melkova Z. Lytic infection with vaccinia virus activates caspases in a Bcl-2-inhibitable manner. Virus Research 2008; 135: 53–63.

Assessment of PI3K and MAPKs roles in expression of ABC transporters performed by dye exclusion assays

Procházková, J.¹, Sotolářová, K.¹, Procházková, J.², Kozubík, A.^1,2, Pacherník, J.¹

¹Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Brno; e-mail: jipro@sci.muni.cz

²Department of Cytokinetics, Institute of Biophysics, ASCR, v.v.i, Brno

Cancer therapy failure is often caused by selection of clones resistant not only to the chemotherapeutics used, but usually also to other type of drugs. One of participating mechanism of such multidrug resistance (MDR) is overexpression of ATP-dependent transmembrane carriers, so called ABC (ATP-binding cassette) transporters (Gottesman et al. 1995).

There are three main types of MDR associated ABC transporters in mammals – MDR1/ABC B1/Pgp, MRP1/ABC C1, and BCRP/ABC G2 (reviewed in Li et al., 2007). Natural role of these transporters is to protect organism against cytotoxic agents. Their expression is thus increased in cells important for integrity of organism as are stem cells and other less differentiated progenitor cells. Cancer cells often display such undifferentiated or dedifferentiated phenotype, too. Higher ability to exclude dyes is one of major hallmarks of progenitor cells. These rare less stained cells thus create so called “side” population next to the major, intensively stained population of cells.

Recently it was found out that expression of ABC transporters could be in some cases regulated by mitogen activated protein kinases (MAPKs), namely the p38 and ERK subtype (Katayama et al., 2007). In regulation of expression of the ABC transporters is involved also phosphatidyl inositol trisphosphate kinase (PI3K)/ protein kinase B (Akt) pathway (Liang et al., 2009). Those kinases were also shown to modulate the activity of transcription factors involved in regulation of ABC transporters as are p53, AP1, or C/EBPbeta (Scotto 2003). We first aimed to evaluate how is the expression of ABC transporters affected by pharmacological inhibitors of studied kinases (p38, ERK1/2, JNK1/2, Akt) and in the next step we tested the involvement of particular transcription factors.

All the experiments were performed on A549 cell line derived from non small cell lung carcinoma that is rich for all three tested ABC transporters. Dye exclusion assays were tested on leukemic cells transfected by individual types of ABC transporters (HL-60-MDR1, HL-60-MRP1, PLB-BCRP). Calcein AM (0.2 μM) is fluorescent in green spectrum after intracellular cleavage by unspecific esterases and it is a substrate of mainly MRP1 protein. JC 1 (0.25 μg/ml) is a cationic carbocyanin probe that enters mitochondria, emits green light, and is a substrate of MDR1. BCRP protein can exclude a conjugate of FL-1 dye BODIPY and alpha-adrenergic blocker prazosin (0.5 μM) that has the affinity to this ABC transporter. Chlorophyll catabolite pheophorbide A (1 μM) is another substrate of BCRP and it emits in far red channel. For inhibition of dye exclusion and thus testing the specifity of staining we used pharmacologic inhibitors – Cyclosporin D (20 μM, CSD), reported inhibitor of ABC B1/Pgp; Fumitremorgin C (20 μM, FTC), inhibitor of ABCG2/BCRP; MK571 (20 μM, MK886) inhibitor of MRP1, and Verapamil (20 μM, V), general inhibitor of majority of membrane transporters. Staining was performed in 37 °C for 10 minutes (Krejčová et al., 2009). Immunocytochemistry of surface expression or total expression of ABC transporters was performed by FACS or Western blotting, respectively. Used antibodies were produced by Santa Cruz Biotechnol.

Assessment of function of ABC transporters by dye exclusion assays is complicated by the sensitivity of the technique to density of the cell suspension. This obstacle we aimed to overcome by comparison of the sample cells with inner control – cells transfected by particular ABC protein. Transfected cells are significantly less stained than A549 cells as they express extremely high amount of given transporter. Co-incubation of sample A549 cells with ABC transfectant, which we can set to constant median of fluorescence, thus allows a precise comparison.

Dye exclusion assays showed that spontaneous expression of studied ABC transporters is modified by inhibitors of kinases. P38 kinase inhibitor SB203580 (10 μM), JNK inhibitor SP600125 (2 μM) and PI3K inhibitor LY294002 (10 μM) down-regulate expression of all studied ABC transporters (although to a different extent) and thus it is plausible that these kinases stimulate their expression. On the other hand, inhibition of ERK by U0126 (1 μM) hampered cell proliferation and up-regulated ABC transporters expression. The exact mechanism of the U0126 effect is not known yet, but it could be connected to the general defense mechanism of the cell against toxic compounds, which could be mediated by increased expression of ABC transporters (Abolhoda et al., 1999).

Project is supported by GAČR 301/08/0717, AV0Z50040507 and AV0Z50040702 of Academy of Sciences of Czech Republic.

References

1. Abolhoda A, Wilson AE, Ross H, et al. Rapid Activation of MDR1 Gene Expression in Human Metastatic Sarcoma after in Vivo Exposure to Doxorubicin. Clin Can Res 1999; 5: 3352–3356.

2. Gottesman MM, Hrycyna CA, Schoenlein PV, Germann UA, Pastan I. Genetic analysis of the multidrug transporters. Annu Rev Genet 1995; 29: 607–649.

3. Katayama K, Yoshioka S, Tsukahara S, Mitsuhashi J, Sugimoto Y. Inhibition of the mitogen-activated protein kinase pathway results in the down-regulation of P-glycoprotein. Mol Cancer Ther 2007; 6: 2092–2101.

4. Krejčová D, Procházková J, Kubala L, Pacherník J. Modulation of cell proliferation and differentiation of human lung carcinoma cells by the interferon-alpha. Gen Physiol Biophys 2009 (in press).

5. Li F, Tiede B, Massagué J, Kang Y. Beyond tumorigenesis: cancer stem cells in metastasis. Cell Research 2007; 17: 3–14.

6. Liang J, Ge F, Guo Ch. Inhibition of PI3K/Akt partially leads to the inhibition of PrPC-induced drug resistance in gastric cancer cells. FEBS Journal 2009; 276: 685–694.

7. Scotto KW. Transcriptional regulation of ABC drug transporters. Oncogene 2003; 22: 7496–7511.

Flow cytometric methods for description of apoptosis in a context of the cell cycle and the type of the cell death

Vondálová Blanářová O.¹, Jelínková I.^1,2, Souček K.¹, Hofmanová J.¹, Kozubík A.^1,2

¹Department of Cytokinetics, Institute of Biophysics, AS CR, v.v.i, Brno, Czech Republic; e-mail: blanarova@ibp.cz

²Faculty of Science, Masaryk University Brno, Czech Republic

Apoptosis as a programmed form of cell death is an important mechanism for maintaining of homeostasis in the tissues of the multicellular organism. Induction of apoptosis in malignant cells belongs to crucial issues in cancer therapy. There is a need for accurate and non-time-consuming methods for detection of apoptosis in context of other aspects of the cell fate in a development of new anticancer drugs in vitro and in vivo. Flow cytometry fulfil the requirements, because it makes possible to combine detection of various markers in an individual cell.

Induction of apoptosis by many antitumor drugs is strongly dependent on position of the cell in the cell cycle. Description of relation of induction of apoptosis on cell cycle phase may shed light on mechanisms of activation of apoptotic pathways as well as rise of resistance to the anticancer therapy and resistance prevention. TUNEL assay followed by staining of DNA by propidium iodide is a widely used method for deciphering cell cycle phase specific apoptosis. However, detection of DNA breaks is somehow hindered in many cell types and there is a great advantage to use methods for detection apoptosis on different basis than DNA fragmentation. We tested methods based on detection of active form of caspase-3 and presence of cleaved fragments of cytokeratin 18. Cytokeratin 18 is a part of cytoskeleton in epithelial cells and is cleaved in early stages of apoptosis by active effector caspases-3, -6 and -7. They proved to be better suited for detection of apoptosis with analysis of the cell cycle than TUNEL assay in our experimental model of epithelial colon adenocarcinoma cell line HCT-116. Cell cycle specific apoptosis was analysed after incubation with various anticancer cytotoxic drugs.

Another question frequently asked and answered by flow cytometry is time course of apoptotic machinery and decision of the individual cell between apoptosis as a programmed cell death and necrosis, eventually secondary necrosis. We studied method employing cellular probe, which localise only to the death cells, even after permeabilisation of the plasmatic membrane Live/Dead Fixable Dead Cell Stain (Invitrogen). Detection of fluorescence of this probe is very useful for resolution between apoptosis and necrosis with combination of detection of various apoptotic markers, even in fixed samples. We tested this probe in combination with M30 and active caspase-3 antibody and both methods gave straightforward and consistent results.

This work was supported by grants No. 1QS500040507 IGA AS CR and 301/07/1557 GA CR.

Platinum(IV) complex LA-12 modulates expression of trail receptors in human colorectal carcinoma cell line

Jelínková I.^1,2, Vondálová Blanářová O.^1,2, Hofmanová J.^1,2, Sova P.³, Kozubík, A.^1,2

¹Department of Cytokinetics, Institute of Biophysics, AS CR, v.v.i, Brno, Czech Republic; e-mail: ivina@ibp.cz

²Faculty of Science, Masaryk University Brno, Czech Republic

³R&D, Pliva-Lachema, a.s, Brno, Czech Republic

Platinum compounds are anticancer drugs frequently used in the therapy of solid tumours. Recently, platinum(IV) complexes have been introduced as an important approach to the treatment of cancer. Novel promising anticancer adamantylamine Pt(IV) complex LA-12 was found to be highly effective in various cancer cells representing different types of tissues.

TNF-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in a number of tumour cell lines, but not in most normal human cells. Therefore it is an intensively studied promising candidate for cancer therapy. However, some cancer cells are resistant to TRAIL effects. The elucidation of the TRAIL signalling pathways is hence essential for proper understanding of the mechanisms involved in cell resistance at different levels. It is possible to overcome resistance and increase apoptosis, e.g. by combination of TRAIL with platinum compounds. Cisplatin treatment mediated sensitivity to TRAIL-induced cell death in human granulosa tumour cells (Woods, 2008). It has been shown that treatment with cisplatin upregulates TRAIL receptors DR4 and DR5 and sensitizes cells to TRAIL induced apoptosis in esophageal squamous cell carcinoma (Kondo, 2006).

The aim of this study was to describe the effects of LA-12 on sensitivity to TRAIL-induced cell death by upregulation of TRAIL receptors in human colorectal carcinoma cell line HCT116 p53^+/+. Our data showed that pretreatment with LA 12 increase expression of TRAIL receptors on cytoplasmic membrane and subsequent incubation with TRAIL may increase sensitivity to induced apoptosis.

This work was supported by grants No. 1QS500040507 IGA ASCR and 301/07/1557 GACR.

References

1. Kondo K, et al. Prospective antitumor effects of the combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cisplatin against esophageal squamous cell carcinoma. Surg Today 2006; 36: 966–974.

2. Woods DC, Alvarez C, Johnson AL. Cisplatin-mediated sensitivity to TRAIL-induced cell death in human granulosa tumor cells. Gynecol Oncol 2008; 108: 632–640.

1,25-dihydroxyvitamin D3 modulates NSAIDs-induced expression of growth differentiation factor 15

Slavíčková E.^1,2, Lincová E.^1,3, Pernicová Z.^1,3, Staršíchová A.¹, Kozubík A.^1,3, Souček K.¹

¹Department of Cytokinetics, Institute of Biophysics, Brno, Czech Republic; e-mail: slavickova@ibp.cz

²Department of Genetics and Molecular Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

³Department of Animal Physiology and Immunology, Faculty of Science, Masaryk University, Brno, Czech Republic

Anti-tumorigenic effects of vitamin D3 and nonsteroidal anti-inflammatory drugs (NSAIDs) are well established in several types of cancer disease. The potential use of NSAIDs as anticancer drugs through their inhibition of proliferation and induction of apoptosis has been documented. Interestingly it was shown that interaction between effects of NSAIDs and vitamin D3 might exist, but mechanisms driving these processes are still unknown.

TGF-ß proteins are autocrine mediators of several effects of biologically active compounds such as vitamin D3. An important cytokine from TGF-ß family is growth differentiation factor 15 (GDF-15/MIC-1). GDF-15 belongs to stress-associated genes and regulates inflammatory and apoptotic pathways in injured tissues. Its high expression in prostate cancer tissue and serum of the patients suggests a possible role in tumor progression. Prostate cancer is a frequently diagnosed malignancy and is the leading cause of cancer-related deaths in Western world. Therefore, public health implication of identifying an effective chemopreventive strategy and treatment against this disease is very considerable.

In our study, we analyzed effects of indomethacin (non-specific inhibitor of cyclooxygenases) in combination with dihydroxyvitamin D3 on cytokinetics and GDF-15 expression in prostate cancer epithelial cells LNCaP. Our data show that mutual treatment of indomethacin and vitamin D3 inhibits cell proliferation. We observed a significant decrease in cell numbers and induction of cell cycle arrest. Simultaneously, GDF-15 expression was increased on both mRNA and protein level. Using western blotting, we detected both the precursor and the active form of GDF–15. Expression of the active form was significantly increased in samples treated with both indomethacin and vitamin D3 compared to treatments with only one of these drugs. Using ELISA we further confirmed the increased level of active form of GDF-15 in conditioned medium of LNCaP cells after the treament.

In summary our data suggest a possible role of GDF-15 in antiproliferative action of dihydroxyvitamin D3 and NSAIDs. Their ability to induce inhibition of proliferation demonstrates great potential for chemoprevention in several types of cancer.

This work was supported by grant No. 204/07/083 of the Czech Science Foundation, grant No. 9600-4 of the Ministry of Health of the Czech Republic and grants Nos. AV0Z50040507, AV0Z50040702 of the Academy of Sciences of the Czech Republic.

Measurement of viability of adult cardiomyocytes under specific culture conditions

Skopalik J.¹, Matejovič P.², Pásek M.², Scheer P.³, Klabusay M.¹, Stejskal S.⁴

¹Laboratory of Flow Cytometry and Cellular Therapy, Faculty of Medicine, Masaryk University, Brno, Czech Republic; e mail: j.skopalik@seznam.cz

²Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

³Department of Physiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic

⁴Department of Biomedical Imaging, Faculty of Science, Masaryk University, Brno, Czech Republic

Isolated cardiac myocytes from adult rat have been used for electrophysiological and pharmacological experiments for more than twenty years. In order to study mechanism of myocardial regeneration, this type of cells could be used for in vitro “stem cell - myocyte cocultivation experiments“. The cocultivation experiments have been done only with neonatal myocyte yet (Beeres 2005 and others). If we want to start the cocultivation experiments with adult rat myocyte, at first we must analyze myocyte viability and functionality during short-term cultivation in RPMI medium with addition of fetal bovine serum (standard condition for stem cell cultivation).

Rat adult cardiac myocytes were isolated (method published by Bébarová 2005), the cells were then cultured in RPMI medium with fetal bovine serum. Viability, relative systolic shortening, amplitude of intracellular calcium concentration oscillating and long-term changes of cell geometry was measured over 3 days of the culture. The calcein + propidium iodide kit (Invitrogen) was used to viability measurement, the systolic shortening and geometry changes were determined by fluorescence microscope (Axiovert 40 CFL, Zeiss) with digital camera (Sony) and VLC graphic software (Fig. 1). The calcium concentration oscillations were visualized using Ca2+ sensitive fluorescent dye fluo-4 (Invitrogen).

Initial viability of the myocytes ranged from 20 to 50%. The amount of viable cells decreased only very slowly over 3 days of culture. Most of the viable cells preserved rod shape, others became rounded. Relative systolic shortening was about 10% at the beginning of cultivation and about 5% after 48 hours.

We can conclude that significant part of the cultivated myocytes preserved their viability and good functional properties, this type of myocytes is suitable for 24 and 48 hours in vitro “stem cell – myocyte cocultivation experiments“.

References

1. Bébarová M, Matejovič P, Pásek M, Šimurdová M, Šimurda J. Effect of Ajmaline on Action Potential and Ionic Currentsin Rat Ventricular Myocytes. Gen Physiol Biophys 2005; 24: 311–325.

2. Beeres S, Atsma ED, der Laarse A. Human Adult Bone Marrow Mesenchymal Stem Cells Repair Experimental Conduction Block in Rat Cardiomyocyte Cultures. Journal of the American College of Cardiology 2005; 46: 1943–1952.

Flow cytometric detection of a composite low grade B-cell lymphoma with two immunophenotypically distinct cell populations

Stránská E, Veselá E, Kalinová M, Kamarádová K, Manďáková P, Augustiňáková A, Campr V, Kodet R.

Department of Pathology and Molecular Medicine, Charles University in Prague, 2nd Medical Faculty, Prague, Czech Republic; e-mail: eliska.stranska@fnmotol.cz

Composite lymphomas are defined as two morphologically, immunophenotypically and genetically distinct types of lymphoma, either two non-Hodgkin’s lymphomas (NHL), or a rare association of NHL and Hodgkin lymphoma (HL), localized in the same anatomical site (excluding lymphoma progression or transformation). The incidence of these tumors is low.

We present an unusual case of a lymphoma arising in a 57-year-old man with a progressing temporal subcutaneus resistance. After the biopsy was done, the flow cytometric immunophenotyping revealed two distinct CD19⁺ B-cell subpopulations. One of them was composed of small cells (15% from lymphoid gate) with lambda light chain restriction and coexpression of CD5, CD20and CD23 and corresponded to B-cell chronic lymphotic leukemia (B-CLL). The other subpopulation was composed of middle-sized cells (40% from lymphoid gate) with bright expression of lambda light chains and immunophenotype: CD20⁺ CD23⁺/- CD38⁺ CD10^- CD5^- and was consistent with marginal zone lymphoma (MZL).

The cytometrical diagnosis was confirmed by histological and immuno-histochemical examination. In addition, the molecular detection of the rearranged immunoglobulin genes (IgH, IgK and IgL) was performed and showed the biclonal origin of those two phenotypically distinct B-cell subpopulations.

Within the staging of the disease, flow cytometry showed the minimal infiltration of bone marrow with B-CLL only, and bone marrow trepanobiopsy revealed a mild nodular infiltration.

In summary, based on the combination of diagnostic and following clinical examinations, the represented case was classified as a composite lymphoma: cutaneous marginal zone lymphoma (MZL), clinical stage I A, and B-cell chronic lymphotic leukemia (B-CLL), clinical stage IV A.

This work was supported by Research Project FNM MZO 00064203 n. 6704.

References

1. Fend F, Quintanilla-Martinez L, Kumar S, et al. Composite Low Grade B-Cell Lymphomas with Two Immunophenotypically Distinct Cell Populations Are True Biclonal Lymphomas. Am J Pathol 1999; 154: 1857–1866.

2. Mokhtar NM. Composite Lymphoma. J Eg Nat Can Inst 2007; 19: 171–175.

3. Thirumala S, Esposito M, Fuchs A. An Unusual Variant of Composite Lymphoma - A Short Case Report and Review of the Literature. Arch Pathol Lab Med 2000; 124: 1376–1378.

Expression CD27 on plasma cells in monoclonal gammopathies

Kovářová L.^1,2, Burešová I.^1,2, Suská R.^1,2, Zarbochová P.³, Hájek R.^1,2,4

¹Department of Haematology, Laboratory of Experimental Haematology and Cell Immunotherapy, University Hospital, Brno, Czech Republic; e-mail: lkovarova@fnbrno.cz

²University Research Centre-Czech Myeloma Group, Faculty of Medicine, Masaryk University, Brno, Czech Republic

³Faculty of Science, Masaryk University, Brno, Czech Republic

⁴Department of Haemato-oncology, University Hospital and Faculty of Medicine, Masaryk University, Brno, Czech Republic

Flow cytometry is widely used tool for analysis of plasma cells (PCs) in monoclonal gammopathies (MG), although is not necessary for diagnosis of monoclonal gammopathy of undetermined significance (MGUS) and/or multiple myeloma (MM). However PCs phenotyping is useful for differential diagnosis, prediction of outcome and minimal residual disease (MRD) monitoring in MM as well. MM is characterized by a clonal expansion of neoplastic PCs. These PCs are mostly CD19^-CD56⁺, but in a small group of patients clonal CD19⁺ PCs occur. Light chain clonality assessment is widely used to discriminate between clonal and polyclonal PCs, although, especially in low infiltration cases, results are not convincing. CD27 is expressed on memory B-cells and also on normal polyclonal CD19⁺ plasma cells, so one can assume that this marker can be used for confirmation of polyclonal CD19⁺ PCs in MM as well.

The aim of this study was to analyze expression of CD27 on CD19⁺ PCs in different groups of MG patients and to prove that CD27 can be routinely used to confirm polyclonality of CD19⁺ PCs.

A total of 60 patients were analyzed and this group consisted of 15 MGUS patient; 10 newly diagnosed MM patient; 9 MM patient with possibility of relapse; 14 MM patient after transplantation; and 10 control patient without MGUS and/or MM. Bone marrow was incubated with appropriate monoclonal antibodies (Immunotech, Beckman Coulter), lysed and analyzed by flow cytometer FC500 (Beckman Coulter). PCs were identified as CD38⁺CD138⁺ cells and expression of CD19, CD56, and CD27 was analyzed on these PCs. Flow cytometry analysis of clonality assessment using kappa and lambda light chains was done in almost all cases.

When analyzed MGUS patients, correlation between medians of CD19⁺ PCs (37.1%) and CD19⁺CD27⁺ PCs (40.9%) was found (0.95). Only 1 case with clonal CD19⁺ PCs was found in new MM; median of CD19⁺ PCs (0.9%) and CD19⁺CD27⁺ PCs (2.0%) was low, although correlation was higher than in MGUS (0.97). There were found 3 cases of clonal CD19⁺ PCs in subgroup of MM patients with possible relapse, median of CD19⁺ PCs (54.0%) was higher than CD19⁺CD27⁺ PCs (42.2%) and correlation was lower than in previous subgroups (0.87). There was found only 1 case of clonal CD19⁺ PCs in MM patients after transplantation, thus correlation between CD19⁺ PCs (78.5%) and CD19⁺CD27⁺ PCs (80.8%) was similar to MGUS and new MM subgroups (0.92). As was expected, high correlation between CD19⁺ PCs (63.8%) and CD19⁺CD27⁺ PCs (64.7%) was found in patients without MGUS/MM (0.94). Finally, the correlation between median of CD19⁺ PCs and CD19⁺CD27⁺ PCs in group of 5 patients with clonal CD19⁺ PCs was significantly lower (0.36) than in group 55 patients with phenotypically polyclonal CD19⁺ PCs (0.96).

We confirmed that CD19⁺CD27⁺ PC are phenotypically normal polyclonal PCs and CD19⁺CD27^- PCs are clonal PCs in all cases. In our hands expression of CD27 on CD19⁺ PCs can discriminate between clonal and polyclonal CD19⁺ PCs and thus can be potentially used instead of labor intensive light chains assessment.

This work was supported by LC06027, GACR 301/09/P457 and MSM0021622434.

References

1. Bataille R, Jego G, Robillard N, et al. The phenotype of normal, reactive and malignant plasma cells. Identification of “many and multiple myelomas” and of new targets for myeloma therapy. Haematologica 2006; 91: 1234–1240.

2. Guikema JE, Hovenga S, Vellenga E, et al. CD27 is heterogeneously expressed in multiple myeloma: low CD27 expression in patients with high-risk disease. British Journal of Haematology 2003; 121: 36–43.

3. Rawstron AC, Orfao A, Beksac M, et al. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders. Haematologica 2008; 93: 431–438.

Elevated regulatory T cells (Tregs) in the blood of psoriatic patiens treated with Goeckerman’s therapy

Kondelkova K.¹, Vokurkova D.¹, Borska L.², Fiala Z.³, Hamakova K.⁴, Rehacek V.⁵

¹Institute of Clinical Immunology and Allergology, Faculty of Medicine in Hradec Králové, Charles University in Prague, University Hospital, Hradec Králové, Czech Republic; e-mail: Katerina.Kondelkova@seznam.cz

²Institute of Pathological Physiology, Faculty of Medicine in Hradec Králové, Charles University in Prague, Czech Republic

³Institute of Hygiene and Preventive Medicine, Faculty of Medicine in Hradec Králové, Charles University in Prague, Czech Republic

⁴Clinic of Dermal and Venereal Diseases, University Hospital, Hradec Králové, Czech Republic

⁵Transfusion Department, University Hospital, Hradec Králové, Czech Republic

Regulatory T cells (Tregs) are a specialized subpopulation of T cells that act to suppress immune responses, thereby maintaining homeostasis and self-tolerance (Liu et al., 2006). It has been shown that Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity (Grant et al., 2009). Psoriasis, a common disorder affecting 1–2% of individuals in Western societies, bears many features of a T cell-mediatedautoimmune disease. Goeckerman’s therapy (GT) of psoriasis is based on a daily application of pharmacy coal tar on the affected skin with subsequent exposure to UV light (Borska et al., 2007). The purpose of this study was to compare the quantity of Tregs in the peripheral blood between psoriatic patients and healthy volunteers and then to evaluate the influence of GT on the frequency of Tregs in the peripheral blood of patients with psoriasis.

26 patients with chronic plaque form of psoriasis (11 males, 15 females, average age 45.8 years, range 18–81 years) and 20 healthyvolunteers (14 males, 6 females, average age 39.8 years, range 22–62 years) were enrolled in the study. Psoriasis areas and the severity index were measured (PASI score 17.5 ± 8.8). The immunophenotypic analysis was performed on erythrocyte lysed heparinized peripheral blood on a flow cytometer FC500 Cytomics (Beckman Coulter) with a 4-color antibody panel: CD3-FITC/CD127-PE/CD25-PC5/CD4-PC7. Appropriate isotype-matched negative control was used to assess the background fluorescence intensity. The obtained data were analyzed using software CXP Analysis. Tregs were characterized by the expression of CD3⁺CD4⁺ CD25^highCD127^low/- phenotype.

Statistical differences between the groups were evaluated by the non-paired and paired t–test (MedCalc software) after data normality evaluation. The results are given as the mean ± standard deviation. P value less than 0.05 is recognized as significant. Tregs were significantly higher (P = 0.0042) in the patients with psoriasis after GT (4.3 ± 1.6) than at the beginning of the therapy (3.2 ± 1.1). There was no significant correlation of Treg admission in the peripheral blood of the healthy controls and psoriatic patients.

This study describes the changes in the frequency of Tregs in peripheral blood of patients with psoriasis treated with GT. This therapy is highly efficient in the treatment of psoriasis and it achieves good clinical response followed by a long-term remission in a majority of patients. The disease activity was significantly diminished by GT (from 17.5 ± 8.8 to 8.0 ± 6.6 after therapy, P < 0.0001). Both the coal tar and UV light exhibit immunosuppressive activity. We found that CD4⁺ T lymphocyte subpopulation in peripheral blood,phenotypically CD25^highCD127^low/- Treg cells, increases suppressor activity in psoriasis.

This work was supported by Research project, MZO, No. 00179906.

References

1. Borska L, Fiala Z, Krejsek J, Andrys C, Vokurkova D, Hamakova K, Kremláček J, Ettler K. Immunologic changes in TNF-alpha, sE-selectin, sP-selectin, slCAM-1, and IL-8 in pediatric patiens treated for psoriasis with the Goeckerman regiment. Pediatr Dermatol 2007; 24: 607–612.

2. Grant J, Bourcier K, Wallace S, Pan D, Conway A, Seyfert–Margolis V, Wallace PK. Validated Protocol for FoxP3 Reveals Increased Expression in Type 1 Diabetes Patiens. Cytometry Part B (Clinical Cytometry) 2009; 76B: 69–78.

3. 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 J. A. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. Journal of Experimental Medicine 2006; 203: 1701–1711.

Vplyv Imunoglukánu^® na imunitnú odpoveď po intenzívnom fyzickom strese u vrcholových športovcov

Tibenská E.¹, Bobovčák M.²

¹OLD-Medirex a.s, Bratislava, Slovenská republika; e-mail: elena.tibenska@medirex.sk

²OKL-VÚPCH Nová Polianka, Slovenská republika

Je známe, že mierna či primeraná fyzická aktivita pozitívne ovplyňuje imunitný systém. Nadmerná a vyčerpávajúca fyzická záťaž naopak navodzuje imunosupresiu, prinajmenšom v niektorých zložkách prirodzenej a získanej imunitnej odpovede. Fyzický stres u pravidelne trénujucich aktívnych športovcov môže navodiť dlhodobú a chronickú depresiu imunitnej odpovede. Boli pozorované zmeny v počte i aktivite NK-buniek po akútnom a aj dlhodobom fyzickom strese. Podobné zmeny boli zaznamenané i v ďalších subpopuláciách imunokompetentných buniek a taktiež i vo funkčnej aktivite fagocytárneho kompartmentu. V niekoľkých štúdiách bol popísaný benefičný efekt beta glukánov na metabolickú aktivitu fagocytov v súvislosti s fyzickým stresom. V našich predchádzajúcich prácach, v experimentoch in vitro, sme dokázali významný modulačný efekt β(1,3/1,6)-D-glukánu izolovaného z huby Pleurotus ostreatus resp. Imunoglukánu^® na funkčné prejavy imunokompetentných buniek.

Cieľom pilotnej štúdie bolo preukázať zmeny v zastúpení buniek periférnej krvi, v subpopuláciách imunokompetentných buniek, a zmeny v aktivite NK-buniek po akútnej vyčerpávajúcej fyzickej záťaži a po hodine relaxácie u aktívnych vrcholových športovcov s cieľom dokázať predpokladaný protektívny a imunomodulačný efekt Imunoglukánu.

Pilotnej štúdie sa zúčastnilo 20 vrcholových pravidelne trénujúcich športovcov zimných disciplín. Štúdia mala dve fázy: v prvej fáze bola aplikovaná záťaž vo forme stupňovitého bicyklového testu až do vyčerpania. Boli vykonané vyšetrenia nasledovných parametrov: krvný obraz, subpopulácie lymfocytov- v relatívnom i absolútnom počte, s dôrazom na NK bunky, subpopulácie NK buniek CD16±/CD8±/CD56± a NK aktivita. Parametre boli vyšetrované pred záťažou, 5 minút po záťaži a po hodinovej relaxácii. Športovci boli rozdelení na dve terapeutické skupiny. Jedna skupina užívala 100 mg Imunoglukánu denne, druhá skupina placebo počas doby dvoch mesiacov pri súčasne prebiehajúcej letnej tréningovej príprave. V druhej fáze štúdie bol aplikovaný rovnaký záťažový test a schéma vyšetrení ako v prvej fáze.

Diferenciálny počet leukocytov a subpopulácie lymfocytov boli vyšetrené bezprostredne po odbere na prietokovom cytometri FC500 (Beckmann-Coulter) po ofarbení monoklonovými protilátkami (Immunotech, Coulter) v štvorfarebnom fluorescenčnom prokole s nasledovným zložením: CD4+CD19-FITC/CD8+CD16+CD56-PE /CD3-PC5/ CD45-PC7. Farbenie: koktail saturačných koncentrácií protilátok v PBS s NaN₃ bol zmiešaný s plnou krvou a inkubovaný v tme 15 minút pri RT, erytrocyty boli lyzované 15 minút lýzou roztokom Versalyse (Immunotech) a vzorka bezprostredne analyzovaná na prietokovom cytometri. Absolútne počty jednotlivých populácií buniek boli získané po prepočte na počet leukocytov z vyšetreného krvného obrazu.

Testy na NK aktivitu boli vykonané do 4 hodín po odbere. Izolácia mononukleárnej frakcie leukocytov zo vzorky bola vykonaná mixér flotačnou technikou s použitím Optiprepu (Axis–Shield) podľa odporúčaneho protokolu. NK test bol urobený s použitím firemného flow cytometrického testu: NKTEST – ORPEGEN-Pharma, podľa firemného návodu. Výsledok NK aktivity bol stanovený v lytických jednotkách LU, teda počte efektorov potrebných na 20% lýzu 10 000 terčových buniek a vyjadrený ako počet LU v obsiahnutých v 107 buniek (LU20/107 efektorov). Hodnota bola vypočítaná extrapoláciou z krivky zostrojenej zo všetkých štyroch vyšetrených riedení vzorky pacienta.

V oboch fázach štúdie bola preukázaná vysoko signifikatná zmena (p < 0,00001) v relatívnom a nárast v absolútnom počte imunokompetentných buniek pod vplyvom záťaže a to najmä u NK-buniek. Signifikatný nárast (p < 0,0001) bol pozorovaný aj v hodnotách NK aktivity. Po perióde relaxácie došlo k významnému poklesu relatívneho i absolútneho počtu NK-buniek, a tiež NK aktivity pod bazálnu úroveň. Na pozorované zmeny v porovnaní prvej a druhej fázy štúdie nemala terapia glukánom žiadny vplyv okrem protektívneho efektu na NK aktivitu. V prvej fáze štúdie, NK aktivita poklesla po hodinovej relaxácii u oboch terapeutických skupín pod bazálnu úroveň. V druhej fáze po terapii glukánom sme už signifikatný pokles aktivity u tejto skupiny nezaznamenali, zatiaľ čo u skupiny užívajúcej placebo tento efekt pretrvával.

Naše výsledky preukázali, že počas intenzívneho opakovaného cvičenia dochádza k mobilizácii a redistribúcii buniek imunitného systému v súvislosti so stresom, ktoré môžu viesť k nežiadúcemu efektu depresie imunitnej odpovede. Z pozorovaných údajov usudzujeme, že niektoré nežiadúce efekty najmä v oblasti funkcie buniek je možné modulovať. Práve v tejto oblasti sa ako sľubným ukazuje pozitívny protektívny vplyv partikulárneho fungálneho β(1,3/1,6)-D-glukánu resp. Imunoglukánu^®, na aktivitu NK buniek. Hoci táto malá pilotná štúdia je limitovaná nízkym počtom probandov, jej výsledky naznačujú, že zložitej téme stresovej modulácie imunitnej odpovede je aj v budúcnosti žiadúce venovať pozornosť v rozsiahlejšej a komplexnejšej štúdii zameranej predovšetkým na funkčné prejavy buniek.

Note of the editor: The authors did not submit an English version of the abstract.

EXCELLYSE I - human peripheral blood lysing solution for Becton Dickinson flow cytometers

Benko M.¹, Kalina T.², Havranová M.³, Špryngar M.¹, Škrob F.¹

¹EXBIO Praha, a.s, Prague, Czech Republic; e-mail: benko@exbio.cz

²CLIP Childhood Leukaemia Investigation Prague, Faculty Hospital Motol, Czech Republic

³IMMUNIA, Prague, Czech Republic

Flow cytometry analysis is among the most precise methods used for peripheral blood cell defining and counting so far. Thus, it is of great importance preparing a sample, where any interfering and ineligible cells are removed. So far, Ficoll-Hypaque density gradient method was used to separate leukocytes from whole blood. This method is rather time consuming and may lead to certain leukocyte subset loss. Direct blood sample staining followed by red blood cell lysis therefore takes place instead of density gradient, as a fast and accurate method for whole blood flow cytometry analysis. Antibody stained peripheral blood cells were surface fixed due to the presence of formaldehyde under gentle conditions in buffered solution. Subsequent addition of deionized water created osmotic shock leading to erythrocyte lysis, while preserving leukocytes. Both lyse/wash and lyse/no wash lysing protocols were tested.

Results show that using EXCELLYSE I lysing solution leads to a significant erythrocyte lysis, fixation of leukocytes and antigen preserving. Leukocyte populations are homogenous, well distinguished with very low debris left (as shown in Fig. 1) and without considerable size change. EXCELLYSE I lysing solution shows no significant fluorescence signal quenching of small organic molecules such as FITC, nor large proteins like R-phycoerythrin. Time-dependent, long-term study focused on the stability of the reagent revealed no change in function and quality throughout the whole experiment. No change in reagent’s pH value occurred during the study, a feature related to formaldehyde stability, otherwise formic acid would form as a result of formaldehyde oxidation.

These results demonstrate that EXCELLYSE I is a useful tool for flow cytometry sample preparation when used with BD cytometers.

This work was supported by Exbio Praha, a.s, Prague, Czech Republic.

References

1. Carter PH, Resto-Ruiz S, Washington GC, Ethridge S, Palini A, Vogt R, Waxdal M, Fleisher T, Noguchi PD, Marti GE. Flow cytometric analysis of whole blood lysis, three anticoagulants, and five cell preparations. Cytometry Wiley-Liss, Inc, 1992; 13: 68–74.

2. Tamul KR, Schmitz JL, Kane K, Folds JD. Comparison of the effects of Ficoll-Hypaque separation and whole blood lysis on results of immunophenotypic analysis of blood and bone marrow samples from patients with hematologic malignancies. Clin Diagn Lab Immunol. 1995; 2: 337–342.

The level of CD20 and CD52 antigen expression in minimal residual disease population in patients with chronic lymphocytic leukemia in complete remission

Pevná M, Brychtová Y, Mayer J, Klabusay M.

Laboratory of Flow Cytometry and Cellular Therapy, Faculty of Medicine, Masaryk University, Brno, Czech Republic; e-mail: 22524@mail.muni.cz

Despite significant progress in treatment, chronic lymphocytic leukaemia (CLL) remains incurable disease. Aim of the therapy in most patients is to achieve a complete remission (CR) of disease, which is associated with extension of time to progression and with improved quality of life. Methods of flow cytometry and polymerase chain reaction (PCR) enable to detect minimal residual disease (MRD) in patients with CLL, who achieved CR. CR with negative MRD is associated with favorable extension of the remission period (Bosch, 2008). In some approaches to achieve MRD-negative CR in patients with CLL, monoclonal antibody alemtuzumab (anti-CD52 antibody) is used for consolidation after chemotherapy (O’Brien, 2003) or alemtuzumab therapy is extended until the eradication of MRD is reached (Moreton, 2005). We present a group of patients with CLL, who achieved MRD-positive CR after standard therapy and in whom the level of CD52 antigen expression on residual population of CD5⁺19⁺ cells was determined with the quantitative flow cytometry approach.

Samples of peripheral blood of patients with CLL, who achieved CR after previous treatment according to the NCI-WG criteria („National Cancer Institute - Sponsored Working Group“), were analyzed by flow cytometry. The level of expression of CD20 and CD52 antigens were measured on tumor population of CD5⁺19⁺ cells using the standardized fluorescent microbeads. The level of expression was calculated in molecules of equivalent soluble fluorochrome units (MESF) for each sample.

In the cohort of patients who achieved MRD-positive CR, the median value of MRD was (5%) of leukocytes. The median value of CD20 expression was (25 000) MESF units and the median CD52 expression was (1 025 000) MESF units. In a comparison group of patients, in whom these values were determined before the therapy, the respective values were as follows: (25 000) MESF for CD20 antigen and (245 000) MESF for CD52 antigen. Thus, the results surprisingly show significantly higher levels of expression of CD52 antigen on MRD population in patients with CLL in CR. The cause of this phenomenon is unknown.

Our results may support the concept of MRD therapy with alemtuzumab for the eradication of MRD. High expression of CD52 antigen on the residual population of tumor cells will have advantage in targeted killing of these cells with monoclonal antibody treatment. Further research will be necessary to clarify this issue.

This work was supported by grant IGA MZ CR No. NR9671-4.

References

1. Bosch F, Ferrer A, Villamor N, González M, Briones J, González-Barca E, Abella E, Gardella S, Escoda L, Pérez-Ceballos E, Asensi A, Sayas MJ, Font L, Altés A, Muntanola A, Bertazzoni P, Rozman M, Aymerich M, Giné E, Montserrat E. Fludarabine, cyclophosphamide, and mitoxantrone as initial therapy of chronic lymphocytic leukemia: high response rate and disease eradication. Clin Cancer Res 2008; 14: 155–161.

2. Moreton P, Kennedy B, Lucas G, Leach M, Rassam SMB, Haynes A, Tighe J, Oscier D, Fegan Ch, Rawstrone A, Hillmen P. Eradication of minimal residual disease in B-cell chronic lymphocytic leukemia after alemtuzumab therapy is associated with prolonged survival. J Clin Oncol 2005; 23: 2971–2979.

3. O’Brien SM, Kantarjian HM, Thomas DA, Cortes J, Giles FJ, Wierda WG, Koller ChA, Ferrajoli A, Browning M, Lerner S, Albitar M, Keating MJ. Alemtuzumab as treatment for residual disease after chemotherapy in patients with chronic lymphocytic leukemia. Cancer 2003; 98: 2657–2663.

Long-time follow-up of CD4⁺ T cells in peripheral blood after autologous hematopoietic stem cells transplantation in multiple sclerosis patients

Marečková, H.¹, Krasulová, E.², Havrdová, E.², Kovářová, I.², Trněný, M.³, Vacková B.³, Pohlreich D.³, Kozák T.⁴, Lhotáková P.⁴, Šterzl I.¹

¹Inst.of Immunology and Microbiology, First Faculty of Medicine, Charles University and General Teaching Hospital Prague, Czech Republic; e-mail: hmare@lf1.cuni.cz

²Dept. of Neurology, First Faculty of Medicine, Charles University and General Teaching Hospital Prague, Czech Republic

³Dept. of Haematooncology, First Faculty of Medicine, Charles University and General Teaching Hospital Prague, Czech Republic

⁴Dept. of Clinical Haematology, Third Faculty of Medicine, Charles University, Prague, Czech Republic

High-dose immunoablation with autologous hematopoietic stem cells transplantation (ASCT) presents a treatment with well-known potential of inducing obvious and long-lasting effect on a wide range of autoimmune disorders including multiple sclerosis (MS). Exact mechanisms of ASCT remain largely unknown. First year after ASCT profound lymphopenia can be observed, reconstitution of the particular lymphocyte subsets is however different. Namely delayed CD4⁺ T-lymphocytes recovery has been consistently reported within several studies. The aim of the study was to report the results from long-time follow-up of CD4⁺ T-lymphocytes subsets after ASCT in MS patients.

Eight patients (F/M = 5/3, age 30 ± 8 years, MS duration 7 ± 2 years, EDSS 6.5) with secondary progressive MS underwent ASCT after detailed discussion and informed consent signing. Stem cells were mobilized by high-dose cyclophosphamide and granulocyte colony-stimulating factor, ex vivo purging of the graft was performed, BEAM regime (BCNU, etoposid, cytarabin, melphalan) was used for immunoablation followed by antithymocyte globulin administration intravenously. Peripheral blood was taken and immunophenotyping was performed before ASCT and consecutively 5–11 years after ASCT using flow cytometer FACSCalibur and monoclonal antibodies BD Bioscience (CD4, CD8). MS treatment after ASCT included interferon beta 1b (n = 1), intravenous immunoglobulins (n = 5), azathioprine (n = 2), mycophenolate mofetile (n = 1), cyclosporine A (n = 1), low-dose corticosteroids up to equivalent of 10 mg of prednisolone daily (n = 8). CD4:CD8 ratio exceeded 1 (1.11–1.87) in all patients before ASCT. The treatment led to expected decrease of CD4+ lymphocytes, within 24 months after ASCT CD4:CD8 ratio varied between 0.46 and 1.3. Even 5–11 years after ASCT CD4:CD8 ratio remained lower compared to values before ASCT in every individual patient. Despite a small group of the patients we consistently observed prolonged (5–11 years) recovery of CD4⁺ T-lymphocytes after ASCT in every individual patient. This cannot be referred only to the immunosuppressive or immunomodulatroy treatment after ASCT. Our results document long-lasting ASCT ability to influence the immune system regulation.

The study was supported from the Czech Ministry of Education (research program MSM 0021620807) and from the Czech Ministry of Health (grant IGA MZ CR NR/9375-3/2007).

Detection of activated NK cells in peripheral blood of women with recurrent pregnancy abortions and infertility of unknown aetiology

Vokurková D, Jankovičová K, Burešová E.

Institute of Clinical Immunology and Allergology, Faculty of Medicine and University Hospital in Hradec Kralové, Charles University in Prague, Czech Republic; e-mail: vokurkovad@lfhk.cuni.cz

The regulation of natural killer (NK) cells in the peripheral blood and endometrial layers has been associated with reproductive immunopathology such as recurrent spontaneous abortions (RSA) and infertility of implantation failures (Rai et al., 2005). In addition, immunophenotypic characteristics of NK cells in these women support the changes for their increased activity status. It has been reported that intracellular Th1 cytokine expressions are increased over Th2 cytokine expressions in the peripheral blood of these women (Kwak-Kim and Gilman-Sachs, 2008).

The aim of this study was to investigate the functional status and immunophenotypic characteristics of NK in women who suffer from RSA or infertility of unknown etiology. We tried to set up a fast and simple method of evaluating the activity of NK cells by the detection of the early activation marker CD69 after JAR human trofoblast cell line and sperm cells antigens co-culture, respectively, with the peripheral blood of the women.

Peripheral heparinized blood samples of the infertile women were incubated in 96-well plate tissue culture at 37°C in 5% CO₂ humidified incubator together with tissue culture cell medium for 40 hours. The trophoblast cell line JAR or sperm cell antigens from relevant sexual partner, respectively, were added to the blood samples in optimal concentration. The negative (tissue culture cell medium alone) and positive (pokeweed mitogen) control were enrolled in each experiment. After the incubation, activated NK cells were identified by the detection of CD3, CD56⁺, and CD69⁺ markers. Appropriate isotype-matched negative control was used to assess background fluorescence intensity. Measurement and analysis of the samples were accomplished using the FC500 Cytomics flow cytometer and obtained data acquisition were analyzed using software CXP Analysis (both Beckman Coulter).

In conclusion, peripheral blood NK cells of some women with RSA and infertility of unknown aetiology havehigher proportions of activated NK cells in vivo. UnbalancedCD69 expression may explain the underlying patology (Ntrivalas et al., 2001). We have possibility to compare the reactivity in both co-culture assays with results of the inhibition migration test performed at our department; nevertheless, some women have reactivity in only one of these two tests. However, the first findings indicate that co-culture experiments seems to be another effective assays to investigate cell mediated reactivity in women with reproductive failure.

References

1. Kwak-Kim J, Gilman-Sachs A. Clinical Implication of Natural Killer Cells and Reproduction. Am J Reprod Immunol 2008; 59: 388–400.

2. Ntrivalas EI, Kwak-Kim JY, Gilman-Sachs A, Chung-Bang H, Ng SC, Beaman KD, Mantouvalos HP, Beer AE. Status of peripheral blood natural killer cells in women with recurrent spontaneous abortions and infertility of unknown aetiology. Hum Reprod 2001; 16: 855–861.

3. Rai R, Sacks G, Trew G. Natural killer cells and reproductive failure-theory, practice and prejudice. Human Reprod 2005; 20: 1123–1126.

Expression of RAN, ZHX-2 and CHC1L gene in multiple myeloma

Legartová S, Harničarová A, Bártová E, Galiová G.

Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i, Brno, Czech Republic; e-mail: sonicka.sonicka1@centrum.cz

Gene expression studies by the use of Real Time PCR are considered as a powerful tool for analysis of genes involved in pathophysiology of human diseases. Recent studies of RAN (6p21), ZHX-2 (8q24.3), CHC1L (13q14.3) loci show an importance of these gene expression for multiple myeloma (MM) prognosis and progression (Armellini et al., 2008). ZHX 2 gene was observed as a weakly expressed in a high-risk disease, while increased ZHX–2 expression was associated with better responses and longer survival after high-dose chemotherapy. On the other hand, CHC1L gene was less expressed in hyperdiploid cells and RAN locus was highly transcriptionally active in symptomatic MM and myeloma cell lines (Armellini et al., 2007). ZHX-2 and CHC1L loci are mapped close to the c-myc and Rb1 genes, which both play an important role in tumor cell proliferation (Avet-Loiseau et al, 2001; Shaughnessy et al., 2000); therefore, these genomic regions represent important prognostic markers for many tumors.

In our laboratory we tried to established Real Time PCR methodology for RAN, ZHX-2 and CHC1L genes, which could be applied in clinics. We tried to find a relevant internal control for RT-PCR reaction; therefore, the expression of selected genes was studied in peripheral blood cells of healthy individuals and in myeloma cell lines. Our preliminary results showed, that expression of candidate genes should be analyzed in the cells of newly diagnosed patients and expression levels should be compared only in the cell populations with identical infiltration of malignant clone to bone marrow. Additionally, RNA purity and proper RNA parameters obtained by Agilent Bioanalyzer should be set up for routine RT-PCR diagnostics of tumor-related gene expression. Our experiments showed methodological approaches and troubleshooting on how gene expression can be analyzed by clinical laboratories.

This work was supported by Ministry of Education, Youth and Sports of the Czech Republic, Grant No. LC06027, ME919 and following projects: AVOZ50040507, AVOZ50040702.

References

1. Armellini A, Sarasquete ME, García-Sanz R, Chillón MC, Balanzategui A, Alcoceba M, Fuertes M, López R, Hernández JM, Fernández-Calvo J, Sierra M, Megido M, Orfčo A, Gutiérrez NC, González M, San Miguel JF. Low expression of ZHX2, but not RCBTB2 or RAN, is associated with poor outcome in multiple myeloma. Br J Haematol 2008; 141: 212–215.

2. Avet-Loiseau H, Gerson F, Magrangeas F, Minvielle S, Harousseau JL, Bataille R. Rearrangements of the c-myc oncogene are present in 15% of primary human multiple myeloma tumors. Blood 2001; 98: 3082–3086.

3. Shaughnessy, J, Tian, E, Sawyer, J, Bumm, K, Landes, R, Badros, A, Morris, C, Tricot, G, Epstein, J, Barlogie, B. High incidence of chromosome 13 deletion in multiple myeloma detected by multiprobe interphase FISH. Blood 2000; 96: 1505–1511.

Cell cycle analysis by flow cytometry as power tool for identification of potential drugs with anticancer activity

Lachnitova L, Kollareddy M, Kamenickova A, Potockova J, Dzubak P, Hajduch M.

Laboratory of Experimental Medicine, Department of Pediatrics and Oncology Palacký University, Faculty of Medicine and Dentistry, Olomouc, Czech Republic; e-mail: lenka.lachnitova@seznam.cz

Flow cytometry became the helpful tool for the investigation of cellular parameters, such as their volume and morphological complexity, DNA or RNA content, cell surface or intracellular antigens, enzymatic activity and many others. We used flow cytometry to identify potential drugs with anticancer activity. We tested mechanism of anticancer actions of different quinolinone and triterpene derivates on the population of leukemic CEM cells. To estimate the differences in the cell cycle we used four methods – basic analysis of cell cycle and apoptosis by PI staining, monitoring of RNA/DNA synthesis by BrU/BrdU incorporation and analysis of mitotic marker pH3^Ser10.

Monitoring of RNA synthesis is based on incorporation of BrU (5’- bromouridine) into RNA. Following the formaldehyde fixation is BrU detected by the anti-BrdU (anti-5’-bromo-2--deoxyuridin) antibody. Thus we can measure the cellular synthesis of RNA. Similarly, BrdU (5’-bromo-2-deoxyuridine) as an analog of thymidine is incorporated into DNA and visualized by the anti-BrdU antibody. After adding fluorescent PI which intercalate into DNA we can enumerate the fractions of cells in G0/G1, S and G2/M phases of the cell cycle and distinguish diploid and tetraploid cells. Histone H3 is phosphorylated only during the mitosis; therefore the method based on phosphorylation of histone H3 on Ser 10 enables us to discriminate mitotic cells. Increased capacity of our tests is also achieved by use of HTS module and batch analysis in connection with FACSCalibur.

We found that some of the tested quinolinones and triterpenes induced significant changes in the cell cycle, RNA and DNA synthesis of CEM cell population. Some derivatives were able to block the cells in the S phase or during the mitosis. One promising compound was inducing the polyploidy of the cell population suggesting some regulatory mechanisms in the Aurora or Polo – kinase pathways. These interesting compounds are further studied and we will try to find detailed information about their mechanisms of anticancer activities.

Study is supported by grants GACR 301/09/P433, LC07017, MSM6198959216 and EHP/Norway CZ0099.

Incorporation of gene for green fluorescent protein into cancer cells as the first step in study of experimental tumor complexity

Valaskova Z.¹, Vrabcova M.¹, Markovicova D.¹, Lackovicova L.², Mravec B.^1,2, Bizik J.³, Perzelova A.⁴, Macikova I.⁴, Danihel L.⁵, Kinova S.⁶, Buckingham T.¹, Hulin I.¹

¹Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia; e-mail: zuzana.valaskova@fmed.uniba.sk

²Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia

³Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia;

⁴Department of Anatomy, Faculty of Medicine, Comenius University, Bratislava, Slovakia

⁵Institute of Pathological Anatomy, Faculty of Medicine, Comenius University, Bratislava, Slovakia

⁶Ist Department of Internal Medicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia

Understanding of living as a remarkable phenomenon is a great stimulus for progress in the field of scientific research. Proteins represent the essence of life. They participate in every manifestation in nature and form part of each cell. The study of protein in real state and the endeavor to define structural components of cells were limited on fixative samples as in living tissue antibody binding on protein results in changes of function and behavior of this protein. A great progress in the study of dynamic protein processes was the discovery of gene for green fluorescent protein (GFP) for which in October 2008 the Nobel Prize in chemistry was awarded. Primal position as ’a green crayon’ thus is replaced by prominence position in revolution of cell processes imaging techniques.

The process of tumor formation in a living organism is composed of several steps and can be seen as a type of Darwinian evolution on a microscopic level. During the tumor process some aspects cannot be arranged by duration and sequence. That is why the process can be attributed as one with signs of complexity as a characteristic that is happening in time, but it is always a non-linear process.

In our laboratory, for study of complexity of interaction between tumor and organism, induction of tumor process by application of tumor cells BP6 is extensively used (Mravec et al., 2009). However experiments that used application of tumor cell into animals are often limited by detection of tumor process in incipience. Recently in the field of cancer biology a tumor cellular marker – the gene encoding GFP is being used. We tested the ability of BP6 cells transfected with GFP to induce tumor process in Wistar rats (58 total: 25 male and 33 female). Our data suggest interference between BP6 cells and GFP. It was anticipated that incorporation of GFP gene might change physiological properties of cytoskeleton, worsen adhesive characteristics of tumor cells and BP6 cells incorporated with GFP gene have had smaller ability to induce both experimental intraperitoneal and subcutaneous tumors. Study comparing the capability of tumor induction in experimental model of colon adenocarcinoma cells transfected with GFP in immunocompetent and T- and B-cell-deficient SCID mice shows that immune reaction was responsible for early elimination of these tumor cells (Steinbauer et al., 2003). Tumor cells being contacted also by host dendrite cells, macrophages engulfing tumor cells and it’s evident that induction of tumor growth with GFP cells is result of multilevel interactions within organism.

It is more of suspicion than an argument on the enormous significance of described information and partly of the study observations. Because of great importance of protein molecules we can’t repudiate the significance of GFP at the grade of higher regulatory systems than suspension of cell cultures. In view of prospective progress of scientific research we suppose prevalent use of GFP in marking of tumor in organism for host immune system. GFP could enable (supposedly) to monitor proliferation of cells not only within experimental work, but also in human medicine Aftertime GFP could serve as “target” for guide of tumorigenesis inhibiting substances.

This study was supported by the Slovak Research and Development Agency under contract No. APVV-0045-06, VEGA grants (1/3422/06, 1/4251/07, 1/4312/07) and by UK grant 450/2009.

References

1. Mravec B, Lackovicova L, Pirnik Z, Bizik J, Bundzikova J, Hulin I, Kiss A. Brain response to induced peripheral cancer development in rats: dual fos-tyrosine hydroxylase and fos-oxytocin immunohistochemistry. Endocr Regul 2009; 43: 3–11.

2. Steinbauer M, Guba M, Cernaianu G, Köhl G, Cetto M, Kunz-Schughart LA, Geissler EK, Falk W, Jauch KW. GFP-transfected tumor cells are useful in examining early metastasis in vivo, but immune reaction precludes long-term tumor development studies in immunocompetent mice. – Clin Exp Metastasis 2003; 20: 135–141.

Fine needle aspiration biopsy as a new method for the monitoring tumor environment after the treatment with polymeric drugs

Betka J.^1,2, Hovorka O.¹, Větvička D.¹, Bouček J.^1,2, Ulbrich K.³, Říhová B.¹

¹Institute of Microbiology ASCR v.v.i, Prague, Czech Republic; e-mail: yucatan@volny.cz

²Charles University in Prague, 1st Faculty of Medicine Department of Otorhinolaryngology and Head and Neck Surgery, Faculty Hospital Motol, Postgraduate Medical School, Prague, Czech Republic

³Institute of Macromolecular Chemistry ASCR v.v.i, Prague, Czech Republic

Conventional chemotherapy exposes both normal and malignant cells to identical doses of cytotoxic agents and relies upon the enhanced sensitivity of rapidly dividing cancer cells to achieve preferential killing. Water soluble polymers based on N-(2-hydroxypropyl)-methacrylamide (HPMA) bearing anticancer drug doxorubicin bound through amide (DOX-HPMA^AM) or hydrazone (DOX-HPMA^HYD), were developed to eliminate unwanted side effects of conventional chemotherapy. However, the mechanism of activity of the polymeric conjugates and their effect on immune system is far from being understood. Fine needle aspiration biopsy (FNAB) from the malignant suspicious organs in the human body like lymph node or salivary and thyroid gland – is commonly used clinical method, how to acquire the samples from the body with minimal influence to tissues. We have applied this method to study the effect of polymeric drug delivery systems on the immune system.

C57BL/6 mice bearing EL4 T-lymphoma stably expressing Enhanced Green Fluorescent Protein (EL4-EGFP⁺) were treated with DOX-HPMA^AM, DOX-HPMA^HYD or free doxorubicin. FNAB method was applied to isolate samples from both treated and untreated EL4-EGFP⁺ tumors in different time intervals. All samples were stained with specific fluorescently labeled antibodies (anti - CD3, CD4, CD8, CD19, CD25, CD45, CD69 and NK1.1), analyzed by flow cytometry (LSRII, BD) and verified with the samples obtained from the tumor during the autopsy. The same procedure was applied to the samples of different lymph nodes to observe the infiltration of tumor cells.

This method allows us to compare changes in representation and activation of the tumor infiltrating lymphocytes before and during the treatment procedure with parent drug and its polymeric counterpart. It seems, that percentage of CD4 and CD8 lymphocytes has increased rapidly after the treatment by DOX-HPMA^AM and DOX--HPMA^HYD conjugates between third and fifth day after the drug has been applied, but has remained at same level in case of free doxorubicin. The percentage of EGFP positive tumor cells in particular lymph nodes has decreased with the distance of the nodes from the tumor.

This research was supported partly by MEYS 1M0505 and by Grant Agency of Academy of the Sciences of the Czech Republic grant # IAA400200702.

References

1. Muzzafar T, Wei EX, Lin P, Medeiros LJ, Jorgensen JL. Flow cytometric immunophenotyping of anaplastic large cell lymphoma. Archives of Pathology & Laboratory Medicine 2009; 133: 49–56.

2. Říhová B, Kovář L, Kovář M, Hovorka O. Cytotoxicity and immunostimulation: double attack on cancer cells with polymeric therapeutics. Trends in Biotechnology 2009; 27: 11–17.

3. Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer G. Immunological aspects of cancer chemotherapy. National Review Immunology 2008; 8: 59–73.

Fluorescence-activated cell sorting for the transplantation of human embryonic stem cell-derived neural precursors after MCAO in rats

Turnovcová K.^1,2, Kozubenko N.^1,2, Kapcalová M.^1,2, Jirák D.³, Burian M.^1,3, Jendelová P.^1,2, Syková E.^1,2

¹Department of Neuroscience, Laboratory of Stem Cells and Tissue Repair, Institute of Experimental Medicine, ASCR, v.v.i, Prague, Czech Republic; e-mail: karolina.turnovcova@biomed.cas.cz

²Department of Neuroscience and Center for Cell Therapy and Tissue Repair, 2nd Medical Faculty, Charles University, Prague, Czech Republic

³MR Spectroscopy, MR Unit, Department of Diagnostic and Interventional Radiology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic

Human embryonic stem cell-derived neural precursors (hESC-NPs) can be used in therapies to restore function following neurological deficit. Neural precursors derived in vitro, however, show broad heterogeneity depending on the cell lineage, the differentiation protocol and the developmental stage. Such heterogeneity can often lead to tumor formation after transplantation in experimental models of stroke. Here, we describe the cell surface markers characterizing our CCTL14 hESC line in subsequent sub-cultures using fluorescence-activated cell sorting (FACS) in combination with an evaluation of graft survival and tumor formation.

Human embryonic stem cells and hESC-derived neural precursors can be characterized by a broad spectrum of surface and intracellular markers. Undifferentiated embryonic stem cells express immature markers such as SSEA4, SSEA3, TRA-1-60, TRA-1-81, nanog, oct3/4 and sox2, which disappear after the induction of differentiation. At the same time, markers of neural stem and progenitor cells start to appear: CD133, SSEA1 (CD15), A2B5, CD29 and CD271 (p75). Further differentiation leads to the expression of CD24 and neural cell adhesion molecule NCAM (CD56) (Pruzsak et al., 2007).

In our experiments, neural differentiation consisted of two main stages. The initial neural differentiation was induced by noggin; the cells were then cultivated and expanded in serum-free induction media for 14 days. These pre-inducted cells were then marked as passage 1 of Neural Progenitors (NPs) and were either kept in culture for another 13 passages or used for terminal differentiation. In our transplantation experiments, the cells did not undergo terminal differentiation because mature neurons do not survive the transplantation procedure and therefore are not suitable for cell therapy. NPs were expanded, and in each subsequent passage the expression of a number of surface and intracellular markers (SSEA4, TRA-1-60, nanog, CD133, SSEA1, CD271, CD24, CD29, NCAM, NF70, nestin, beta-III-tubulin) was evaluated, in addition to the expression of some somatic markers such as HLA-ABC. Simultaneously, NPs from passages 1, 5, 8 and 10 were transplanted into a rat model of transient focal ischemia. Middle cerebral artery occlusion was performed, and one week later, 100 x 10⁵/μl cells were injected in 2.5–3 μl into the striatum. Histological analysis was performed at the end of the experiment (1 month after cell grafting).

Our study revealed that at the early stages of differentiation (P1-P2), the cells were positive for NCAM/CD133, beta-III-tubulin, NF70 and nestin with the strong coexpression of SSEA4, TRA-1-60 and nanog, as well as the expression of CD24. During the next several passages (P3–P5), the expression of SSEA4, TRA-1-60 and CD24 was downregulated, while the expression of the neural markers NCAM, ß-III-tubulin, NF70 and nestin remained the same. SSEA1 appeared only during passages P2-P6. The expression of CD271 remained high up to P9-P10, and then was downregulated, whereas the expression of HLA-ABC was low up to P8, then gradually increased in later passages (P9–P13). Histological evaluation revealed that the transplantation of nanog- and SSEA4-positive NPs (P1) led to tumor formation in 100% of the recipient rats. A 50% decrease in tumor formation was observed after the transplantation of P5 NPs, compared to P1 NPs. The difference between these populations of cells consists in the complete down-regulation of the expression of the main pluripotent markers (nanog, SSEA-4, TRA-1-60). The transplantation of P8 NPs yielded the best results among all the experimental groups: no tumors were found during 3 months observation after transplantation, and in most grafts (5 of 7 recipients) the cells migrated towards the lesion. Immunohistochemical analysis revealed the co-staining of transplanted cells for HuNu/nestin and HuNu/NCAM, confirming that P8 NPs maintained a neural profile. The differences in the in vitro characteristics of the P8 NP population from the P5 NPs consist of a 4-fold decrease in CD24 and SSEA-1 expression and a 2-fold increase in CD133 expression. The transplantation of P10 NPs was completely safe in terms of tumor formation, but the survival of the grafted cells was much lower (only 5 of 8 grafts survived). P10 NPs did not proliferate, and very weak migration was found in all grafts. Their surface marker expression profile did not reveal significant differences from that of P8 NPs, only the expression of CD271 was lower by 50% and the expression of HLA-I reached a high level (88.5%).

Our results show that the population of hESC-derived NPs that is appropriate and safe for in vivo use has a CD133^hi/CD24^lo/CD15^lo expression profile. However, since the role of these proteins in neurogenesis, the development of the human nervous system and tumor formation is still unclear, further research into neural marker expression profiles should yield new insights relevant to future strategies for cell-mediated therapies.

Support was provided by grants AV0Z50390703, 1M0538, 309/08/H079, LC554 and the EC FP6 project STEMS (LSHB-CT-2006-037328).

References

1. Pruszak J, Sonntag KC, Aung MH, Sanchez-Pernaute R, Isacson O. Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cells 2007; 25: 2257–2268.

CSFE-based proliferative assay in rabbits – preliminary study

Jeklová E.¹, Ondráčková P.¹, Levá L.¹, Šinkora J.², Faldyna M.¹

¹Veterinary research institute, Brno, Czech Republic; e-mail: jeklova@vri.cz

²BD Biosciences, Praha, Czech Republic

The ability of lymphocytes to respond to plant mitogens by their proliferation provides a simple, semiquantitative in vitro correlate of cell-mediated immunity. Various lymphocyte subsets respond differently to lectin stimulation. Generally, concanavalin A (conA), which is most often used as a plant mitogen, preferentially stimulates T cells. Nevertheless, there exist species specific differences in lymphocyte subsets stimulated by various lectins. In humans after conA mitogenic stimulation, mainly CD4⁺ and less CD8⁺ lymphocyte subsets proliferate (Azzolina et al., 1990). The predominant subsets of porcine lymphocytes responding to the above mentioned plant lectin stimulation are CD4⁺CD8⁺, CD8⁺ and γδ TCR⁺ (Dorn et al., 2002). In contrast to that, conA causes proliferation of CD4⁺, CD8⁺, γδ TCR⁺ and noticeable proliferation of B-cells in cattle (Quade and Roth, 1999). While subset responsiveness to specific mitogens has been described for human, pig and cattle lymphocytes, there is little or no published information describing the subsets of lymphocytes that respond to conA or other mitogens in rabbits, which represent animal species often used as an experimental model in human and veterinary research. It is known from earlier studies in rabbits that conA only stimulates proliferation of T lymphocytes (Fanger et al., 1974). Watkins et al. (1984) noted that rabbit CD4⁺ lymphocyte subpopulations are highly responsive to conA mitogen, but there is no information about other lymphocyte subsets.

The aim of this study was to characterize lymphocyte subpopulations that are stimulated by various plant mitogens in rabbits. Using flow cytometry and multicolor immunophenotyping of carboxyfluorescein (CSFE) stained cells we found that, similar to humans, mainly T-cells are stimulated by conA and phytohaemagglutinin in rabbits while pokeweed mitogen represents a predominant B-cell stimulator.

This work was supported by Czech Science Foundation (GA ČR 524/08/P568).

References

1. Azzolina LS, Stevanoni G, Tommasi M, Tridente G. Phenotypic analysis of human peripheral blood lymphocytes by automatic sampling flow cytometry after stimulation with mitogens or allogeneic cells. Ric Clin Lab 1990; 20: 209–216.

2. Dorn AD, Waters WR, Byers VM, Pesch BA, Wannemuehler MJ. Characterization of mitogen-stimulated porcine lymphocytes using a stable fluorescent dye (PKH2) and multicolor flow cytometry. Vet Immunol Immunopathol 2002; 87: 1–10.

3. Fanger MW, Reese AC, Schoenberg MD, Stavitsky AB, Reese AL. Evidence for T lymphocyte subpopulation in the rabbit. J Immunol 1974; 112: 1971–1980.

4. Quade MJ, Roth JA. Dual-color flow cytometric analysis of phenotype, activation marker expression, and proliferation of mitogen-stimulated bovine lymphocyte subsets. Vet Immunol Immunopathol 1999; 67: 33–45.

5. Watkins JR, McNicholas JM, Loken MR, Knight KL. Characterization of functionally distinct subpopulations of rabbit T lymphocytes. Immunology 1984; 53: 659–667.

Spontaneous acute leukaemia in a pet rat (Ratus norvegicus)

Jeklová E.¹, Jekl V.², Ondráčková P.¹, Řeháková K.², Knotek Z.², Faldyna M.¹

¹Veterinary research institute, Brno, Czech Republic; e-mail: jeklova@vri.cz

²University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic

A fourteen month old male pet rat (Ratus norvegicus) was presented to the Avian and Exotic Animal Clinic of the University of Veterinary and Pharmaceutical Sciences Brno due to apathy, dyspnoea and subcutaneous mass on the neck and armpit. The owner stated the duration of these signs for two days. Clinical examination revealed weakness, hunched posture, low skin elasticity, enlargement of peripheral lymph nodes and splenomegaly. The rat demonstrated a mild discomfort on abdominal palpation.

Abnormal haematology and plasma biochemistry results included marked leukocytosis with high amount of blasts, relative neutropenia and lymphopenia, elevated levels of AST, ALP and phosphorus. The patient was euthanised due to infaust prognosis.

At necropsy, multiple round lesions 2 mm in diameter within lung parenchyma were observed. Generalised lymphadenopathy and diffuse splenomegaly was confirmed. Samples of peripheral blood, suprascapular lymph node and bone marrow were obtained for immunophenotyping. Using flow cytometry, neoplastic cells showed high forward scatter and in all examined organs, the cells were of uniform phenotype CD8⁺, CD172α⁺, CD161⁺, CD3^-, CD4^-, IgM^-, CD79α^-. Definitive diagnosis was spontaneous acute leukaemia.

This study was supported by Ministry of Agriculture of the Czech Republic (MZe 0002716102).

Leukemic infiltration of central nervous system in dogs with acute lymphoblastic leukemia

Borska P.¹, Faldyna M.², Leva L.², Schanilec P.¹, Blatny J.³, Stourac P.⁴

¹Small Animal Clinic, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic; e-mail: lymfom@yahoo.com

²Veterinary Research Institute, Brno, Czech Republic

³Department of Clinical Hematology, Children’s University Hospital, Brno, Czech Republic

⁴Department of Clinical Neurology, Faculty Hospital, Brno, Czech Republic

Central nervous system (CNS) involvement is identified in almost 5% of children and 10% of adult patients with acute lymphoblastic leukemia (ALL) (Bleyer 1985; Cortes et al., 1995). Leukemic infiltration of CNS has diagnostic, therapeutic and prognostic significance and generally is associated with a poor prognosis (Schinstine et al, 2006). Correct appreciation can be complicated by peripheral blood contamination or nonspecific pleocytosis due to intrathecal administration of cytotoxic agents or infection. Immunophenotypization may be helpful for the detection of malignant clones in cerebrospinal fluid (CSF) and can enhance the accuracy of cytologic examination alone (French et al., 2000; Babusikova and Zeleznikova, 2004). To our knowledge, there is no report concerning flow cytometric analysis of CSF in dogs with lymphoproliferative disorders.

Cerebrospinal fluid specimens were collected by cisternalpuncture from 4 dogs with ALL. All samples were submitted to cytologic examination. Flow cytometry analysis was performed and the phenotypic profiles of malignant cells at different sites (CSF, peripheral blood, bone marrow) of the same patient were compared. The cells were counted in a Fuchs-Rosenthal chamber. Differential cell count was evaluated from slides prepared by cytocentrifuge Cytospin-2 (Shandon Ltd, Astmoor, UK) followed by staining with May-Grünwald, Giemsa-Romanowski.A panel of monoclonal antibodies was used to characterize malignant cells. Data were acquired on a standard FACSCalibur™ flow cytometer (Becton Dickinson, Mountain View, CA) operated by the CELLQuest™ software. In each sample, 40 000 cells were measured and the data were saved in the list mode. Propidium iodide was used to stain DNA in dead and damaged cells and to exclude such events from analysis. The WinMDI™ software was used for data processing. Gating was based on forward angle and right angle scatter signals.

Cytologic examination indicated presence of suspected pathological clone in all 4 dogs with ALL included in our study. The immunophenotype of malignant cells in CSF was established in 2 dogs with B-ALL (CD79α⁺CD21^-), while cellularity was > 5 leu/μl. No changes of phenotypic profiles of specimens from different sites were found.

This study was partly supported by Ministry of Agriculture of the Czech Republic (M.F, L.L.) 0002716202 and FRVS (P.B.) 764/G3/2007.

References

1. Babusikova O, Zeleznikova T. The value of multiparameter flow cytometry of cerebrospinal fluid involved by leukemia/lymphoma cells. Neoplasma 2004; 51: 345–351.

2. Bleyer WA. Central nervous system leukemia. Pediatr Clin North Am 1984; 35: 789.

3. Cortes J, O’Brien SM, Pierce S, Keating MJ, Freireich EJ, Kantarjian HM. The value of high dose systemic chemotherapy and intrathecal therapy for central nervous system prophylaxis in different risk groups of adult acute lymphoblastic leukemia. Blood 1995; 86: 2091–2097.

4. French CA, Dorfman DM, Shaheen G, Cibas ES. Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid: increased sensitivity using flow cytometric analysis. Diagn Cytopathol 2000; 23: 369–374.

5. Schinstine M, Filie AC, Wilson W, Stetler-Stevenson M, Abati A. Detection of malignant hematopoietic cells in cerebral spinal fluid previously diagnosed as atypical or suspicious. Cancer 2006; 108: 157–162.

Phenotypical characterisation of lung, peripheral blood and bone marrow mononuclear phagocyte subpopulations and dynamics of their changes during inflammation induced by Actinobacillus pleuropneumoniae

Zelnickova-Ondrackova P.^1,2, Kucerova Z.¹, Nechvatalova K.¹, Leva L.¹, Faldyna M.^1,2

¹Veterinary Research Institute, Brno, Czech Republic; e-mail: zelnickova@vri.cz

²University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic

The aim of this work was to describe the porcine mononuclear phagocyte (MP) subpopulations, which are involved in infiltration of inflamed tissue under in vivo conditions in pigs.

The in vivo infection with Actinobacillus pleuropneumoniae (APP) was used for induction of inflammatory response in the lung tissue. Cell surface markers CD14, CD163, MHCII and SWC9 were used for the identification of MP by flow cytometry. Together with analysis of MP, which appeared in the inflamed lungs, the changes in MP subpopulations in peripheral blood (PB) and bone marrow (BM) compartments were assessed in order to elicit the possible origin and maturation stages of the infiltrating MP.

The population of MP, which migrated to the inflamed lungs, had CD14⁺ CD163⁺ SWC9^+/- MHCII^+/- phenotype. The population of PB MP, which was elevated in the PB after APP infection, was CD14⁺ CD163⁺ SWC9^- MHCII^- suggesting that PB CD14⁺ CD163⁺ MP give rise to the inflammatory monocytes/macrophages. The SWC9 and MHCII molecules appear on these cells after leaving the PB. In healthy animals, the BM MP precursors were represented by CD14^- CD163^- cells maturating directly into CD14⁺ CD163^-, which are then released to the PB. After infection, a completely different maturation pathway of MP precursors appeared; that was represented by CD14^- CD163^- SWC9^- MHCII^- MP directly switching into CD14⁺ CD163⁺ SWC9^- MHCII^- MP. These cells are then probably released directly to the PB.

In conclusion, the populations of MP, which are involved in the inflammatory response in pigs, were described in this work.

This work was supported by grant MZE 0002716202.

Determination of blood and duodenal immunocompetent cells in diets contaminated with DON and ZEA mycotoxins

Revajová V.¹, Levkut M.¹, Herich R.¹, Ševčíková Z.¹, Slaminková Z.¹, Leng L.², Bořutová R.²

¹Department of Pathological Anatomy, University of Veterinary Medicine, Košice, Slovak Republic; e-mail: revajova@uvm.sk

²Institute of Animal Physiology, Slovak Academy of Sciences, Slovak Republic

Mycotoxins are fungal secondary metabolites toxic to animals. They occur in agricultural products and threaten food safety. The presence of mycotoxins in poultry feeds is a significant factor for financial losses to animal industries. Deoxynivalenol (DON), also known by the colloquial name vomitoxin, as well as zearalenon (ZEA), the most common mycotoxins are produced by Fusarium spp. and are included into type B trichothecenes. Syndromes caused by mycotoxins range from acute mortality to slow growth and reduced reproductive efficiency. Consumption of mycotoxins may also result in impaired immunity and decreased resistance to infectious diseases (Oswald et al., 2005). Understanding of their action is essential to predicting potential deleterious effects they might have on human health.

Numerous studies that have been conducted on host resistance, antibody responses and cell-mediated immunity in animals showed that trichothecenes can be either immunostimulatory or immunosuppressive depending on dose, exposure frequency, and timing of functional immune assay (Bondy and Pestka, 2000).

Low dose trichothecene exposure can increase resistance to certain pathogens, elevate serum IgA levels and initiate rapid and transient upregulation of many immune related genes; the latter effect can be exacerbated by concurrent exposure to inflammagenic stimulus such as lipopolysaccharide. High dose exposure severely injures actively dividing tissues including bone marrow, lymph nodes, spleen, thymus, and intestinal mucosa that can result in immunosuppression as evidenced by depression of circulating blood leukocytes, decreased resistance to pathogens, inhibition of antibody responses to model antigens, and impaired delayed type hypersensitivity responses (Pestka et al., 2004).

Leukocytes, which represent the functional cell repertoire of the immune system, are exquisitely sensitive to trichotecenes. Macrophages, B- and T-cells are all affected. The stimulatory effects of low doses of trichothecenes are related to their ability to induce immune- and inflammation-associated genes. In contrast, it appears that the suppressive effects on leukocyte function are linked with the induction of apoptosis as has been demonstrated in macrophages, T and B cells both in vivo and in vitro (Yang at al., 2000).

Human and animal contamination occurs mainly orally and the toxin must traverse the intestinal epithelial barrier before inducing potential health effects. In chicken, DON seems to be rapidly and efficiently absorbed, most probably from the upper parts of the small intestine; absorption is time and concentration-dependent. A rapid plasma clearance and excretion due to an efficient hepatic or renal first-pass effect, rapid intestinal transit time and intestinal microflora which plays a major role in DON detoxification might explain the relative tolerance of poultry. Dietary concentration greater than 5 mg/kg are necessary to cause detrimental effects. Feed refusal and reduced weight gain were found in chickens when dietary concentration of DON reached 16–20 mg/kg, but chronic intake of Fusarium mycotoxins will adversely affect of layer performance, immune response and metabolism (Awad, 2008).

In our experiment we used flow cytometry for study the effect of diets containing low and high dose of DON and ZEA on white blood cells, phagocytic activity of granulocytes, T and B cell numbers in the peripheral blood and duodenal intraepithelial lymphocytes of Ross 308 hybrid chicken broilers. Both doses caused the decrease of total count of leukocytes and lymphocytes. In spite of higher number of heterophiles, their phagocytic activity was lower in comparison to control chickens. Determination of lymphocyte subpopulations in the peripheral blood showed decrease of T cells values (CD3, CD4, CD8, CD44) and MHC II cells, but increase of IgG cells in both dose, and IgM cells only in group fed with low dose. Duodenal intraepithelial lymphocyte (IEL) subpopulations showed decrease values of CD3 and CD4 cells in groups fed with both contaminated diets. Percentage of CD8 and CD44 in group fed with high dose exceeded the values of control. MHC II cells were significantly improved, and IgA cells were significantly decreased in both experimental groups comparing to controls. Two weeks mycotoxins application showed immunomodulatory effects in chickens.

This work was supported by the Research and Development Support Agency, Slovakia, Grant No. APVV-0399-07, APVV-20-041605, VEGA Slovakia, Grant No, 6173, VEGA Slovakia 1/0044/08 and 1/0609/09.

References

1. Awad WA, Ghareeb K, Bohm J, Razzazi E, Hellweg P, Zentek J. The impact of the Fusarium toxin deoxynivalenol (DON) on poultry. Int J Poult Sci. 7: 827-842. 2008.

2. Bondy GS, Pestka JJ. Immunomodulation by fungal toxins. J Toxicol Environ. Health B Crit Rev 2000; 3: 109–143.

3. Oswald IP, Marin DE, Boubet S, Pinton P, Taranu I, Accensi F. Immunotoxicological risk of mycotoxin for domestic animals in Europe. Food Addit Contam 2005; 22: 354–360.

4. Pestka JJ, Zhou H-R, Moon Y, Chung YJ. Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: unravelling a paradox. Toxicol Lett 2004; 153: 61–73.

5. Yang G, Jarvis BB, Chung Y, Pestka JJ. Apoptosis induction by the satratoxins and other trichothecene mycotoxins: relationship to ERK, p38 MAPK and SAPK/JNK activation. Toxicol Appl Pharmacol 2000; 164: 149–160.

Microgeographic genome size differentiation of the wild barley, Hordeum spontaneum, at “Evolution Canyon”, Israel and multispecies comparison

Doležalová I.¹, Pavlíček T.², Gasmanová N.¹, Nevo E.², Hammerová I.¹, Lebeda A.¹

¹Department of Botany, Faculty of Science, Palacký University, Olomouc-Holice, Czech Republic; e-mail: ivana.dolezalova@upol.cz

²Institute of Evolution, University of Haifa, Mt. Carmel, Israel

We used flow cytometry to examine differences of relative genome size (GS) in wild barley, Hordeum spontaneum, between the opposite slopes of the microsite “Evolution Canyon”, Mt. Carmel, Israel (EC). Samples of wild barley were collected in 2004 and 2006.

In both years, higher GS (significantly higher in 2004) was recorded in the population representing the less insolated, cooler, wetter, and microclimatically less fluctuating “European maquis-like” north-facing slope than in the populations of the opposite, more insolated, warmer, and drier “African savannah-like” south-facing slope. In addition, intraslope GS variability and documented differences between both temporal samples indicate the presence of significant spatiotemporal GS fluctuations at EC, in spite of the interslope physical distance of 100 to 400 m. There might be two explanations for significantly smaller GS in 2006 than in 2004: (i) Year to year different microclimate conditions (expected but not measured) are expressed in differential germinability of different genotypes present in the soil seed bank (Yan et al., 2008). (ii) Rapid retrotransposon dynamics including expansions and contractions (e.g, Shirasu et al., 2000; Schulman and Kalendar, 2005) – if we expect that retrotransposons are responsible for most of GS variability in wild barley – is behind spatio-temporal changes in GS even on the generation scale.

Multispecies comparisons do not conclusively support the earlier proposed positive relationship between GS and drought stress (Bureš et al., 2004). In a contrast to the presently studied wild barley were found significant positive correlation between GS and drought stress in C. siliqua (Bureš et al., 2004). In other tested species (C. persicum, L. peregrinus, O. surinamensis) the GS/drought relationship did not reach statistical significance. However, the significant overall multispecies test and the significant Spearman correlation between overall GS and the level of drought stress, provide some support for the proposed relationship or indicate the presence of other environmental factor(s) partly linked to or overlap with the drought stress gradient (Fig. 1).

This study was supported by the Israel Discount Bank Chair of Evolutionary Biology, Ancell-Teicher Research Foundation for Genetics and Molecular Evolution and MSM6198959215.

References

1. Bureš P, Pavlíček T, Horová L, Nevo E. Microgeographic genome size differentiation of the carob tree, Ceratonia siliqua, at “Evolution Canyon”, Israel. Ann Bot 2004; 93: 1–7.

2. Shirasu K, Schulman AH, Lahaye T, Schulze-Lefert P. A contiguous 66 kb barley DNA sequence provides evidence for reversible genome expansion. Gen Res 2000; 10: 908–915.

3. Schulman AH, Kalendar R. A movable feast: diverse retrotransposns and their contribution to barley genome dynamics. Cyt Gen Res 2005; 110: 598–605.

4. Yan J, Chen GX, Cheng JP, Nevo E, Gutterman Y. Phenotypic variation in caryopsis dormancy and seedling salt tolerance in wild barley, Hordeum spontaneum, from different habitats in Israel. Gen Res Crop Evol 2008; 55: 995–1005.

Optimalization of Arabidopsis thaliana protoplast FACS sorting for gene expression analysis

Libus J.¹, van der Graaff E.², Laux T.²

¹Institute of Experimental Botany, AS CR, v.v.i.; Praha, Czech Republic; e-mail: libus@ueb.cas.cz

²Institut für Biologie III, Universität Freiburg, Schänzlestrasse 1, D 79104 Freiburg I. B, Germany

We have improved the previously published method of isolation of specific cell populations from plant roots for microarray analysis of gene expression. The increased yield of material enabled us to avoid RNA amplification and thereby reduce the risk of artifacts. We have employed the method to get data about root apical meristem function in Arabidopsis thaliana.

References

1. Birnbaum K, Shasha DE, Wang JY, Jung J, Lambert GM, Galbraith DW, Benfey PN. A gene expression map of the Arabidopsis root. Science 2003; 302: 1956–1960.

2. Birnbaum K, Jung JW, Wang JY, Lambert GM, Hirst JA, Galbraith DW, Benfey PN. Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines. Nat Methods 2005; 2: 615–619.

Hybridisation of diploid and tetraploid taxa of Centaurea sect. Jacea: frequency, role of unreduced gametes, and morphological variation

Koutecký P, Štech M, Baďurová T, Košnar J.

University of South Bohemia, Faculty of Science, České Budějovice, Czech Republic; e-mail: kouta@prf.jcu.cz

Polyploidy is considered one of the main evolutionary mechanisms in plants. However, process of formation of polyploids is not fully understood yet. Centaurea sect. Jacea includes diploids (2n = 22) and tetraploids (2n = 44). They are strictly sexual and mainly self-incompatible (though autogamy at very low rate is present). Taxa of the same ploidy level (irrespective whether diploid or tetraploid) hybridise frequently and their hybrids are fertile, while hybridisation between the ploidy levels is rare. Hybrids of some taxa can easily be identified morphologically. These features make Centaurea sect. Jacea suitable model for studies of diploid-tetraploid interactions.

Published results of crossing experiments (Gardou, 1972; Hardy et al., 2001) show that inter-ploidy hybrids are usually triploid, i.e. formed by union of reduced gametes. Very rare occurrence of tetraploid hybrids (by unreduced gametes of diploids) was also detected. Strong reproductive isolation between ploidy levels was also confirmed by flow cytometric screening of three mixed populations in which no hybrid triploid individuals were found (Hardy et al., 2000).

Within ongoing study we focus on frequency of hybridisation between the two ploidy levels and role of unreduced gametes. We combine extensive flow cytometric screening of ploidy levels in wild populations (both mature plants and seeds) and crossing experiments. The first results indicate major role of unreduced gametes that has been overlooked by previous studies.

Screening of both seeds and mature plants confirmed that hybridisation between ploidy levels is rare. In seed screening, probably hybrid seeds (both DNA triploids and DNA tetraploids) were found in several mixed populations with very small frequency less then 0.1%. However, they virtually do not contribute to mature population, since within ca 15 mixed populations studied so far hybrids were found in the only one. All hybrids were DNA tetraploids formed most probably by unreduced gametes of diploid (C. elatior) and reduced gamete of tetraploid (C. jacea). No DNA triploids were found. In addition, single DNA hexaploid individual morphologically corresponding to typical C. jacea was found in another mixed population. It originated probably from union of reduced and unreduced gamete of the same species.

In opposite, in crossing experiments hybrids between diploid and tetraploids are regularly produced. They include both DNA triploids (both gametes reduced) and DNA tetraploids (reduced gamete of the tetraploid + unreduced gamete of the diploid). Single DNA pentaploid (reduced pollen of the diploid + unreduced ovule of the tetraploid) was also found. Control crosses within the ploidy level did only yield progeny of the same ploidy level. Accordingly, no deviation from expected DNA ploidy levels was found during flow cytometric screening of single taxon populations.

Our results show that unreduced gametes are formed at both ploidy levels, though their contribution to reproduction is much higher in diploids. Marked discrepancy in number and ploidy levels of hybrids between crossing experiments and wild populations was found. One of possible explanations is pollen competition that could be important isolation mechanism between different ploidy levels. Therefore, we intend to perform another crossing experiment with mixtures of pollen to test the pollen competition.

Hybrids C. elatior ×C. jacea displays unexpected morphological variation even in the first filial generation. It seems that morphology of the progeny depends on a mother plant and it is shifted towards it instead of being intermediate. The hybrids C. elatior C ×C. jacea ? are of particular interest. They are usually tetraploid and some morphotypes much resemble some other tetraploid taxa, such as C. macroptilon and C. oxylepis. Allopolyploid origin of these taxa through unusual pathway involving hybridisation of diploid (unreduced gametes) with already established tetraploid (reduced gametes) can therefore be hypothesised and ought to be tested.

This work was supported by grants 206/08/1126 from the Grant Agency of the Czech Republic and MSM6007665801 from Ministry of Education of the Czech Republic.

References

1. Gardou Ch. Recherches biosystématiques sur la section Jacea Cass. et quelques sections voisines du genre Cenaurea L. en France et dans les régions limitrophes. Feddes Repert 1972; 83: 311–472.

2. Hardy OJ, Vanderhoeven S, de Loose M, Meerts P. Ecological, morphological and allozymic differentiation between diploid and tetraploid knapweeds (Centaurea jacea) from a contact zone in the Belgian Ardennes. New Phytol 2000; 146: 291–290.

3. Hardy OJ, de Loose M, Vekemans X, Meerts P. Allozyme segregation and inter-cytotype reproductive barriers in the polyploid complex Centaurea jacea. Heredity 2001; 87: 136–145.

Towards Proper Evaluation of Image Analysis Methods Used in Biomedical Research

Svoboda D, Kozubek M, Stejskal S.

Centre for Biomedical Image Analysis, Masaryk University, Brno, Czech Republic; e-mail: svoboda@fi.muni.cz

In recent years, the biomedicine, like the other fields of research, has become more and more tied up with the computer science. The vast majority of measurements is acquired, stored and further evaluated in the instruments controlled by the appropriate computer programs. In optical microscopy such equipment has already become a standard as it is very comfortable and fast to use it. The whole process of observation can be typically split into four main parts:

1. Specimen preparation – In the very beginning, the biomedical specialist has to prepare the specimen that is to be observed. This part is usually the most time consuming.
2. Image transmission – This part of observation is controlled by the microscope. The image of the specimen is transmitted through the optical system of the microscope. Due to some imperfections (chromatic aberrations, blur, uneven illumination, etc.) the signal is affected.
3. Image acquisition – In optical microscopy, the image is typically acquired by CCD camera that is responsible for the image formation. Such an image is stored somewhere in the computer hard drive and conveyed for the further processing.
4. Image analysis – The images of some specimens are submitted to selected image analysis methods. These usually include image restoration, image segmentation and image recognition.

The last step of this process, the image analysis, offers a large variety of image processing methods. It is clear that the results of such methods are crucial to final evaluation and conclusions. That is why each user has to verify whether the selected method is appropriate and whether it performs correctly. For this purpose, we implemented a simulation toolbox [1] called CytoPacq (http://cbia.fi.muni.cz/simulator/) that is capable of simulating the first three steps:

• 3D-CytoGen … generates the digital phantom (specimen preparation)
• 3D-OptiGen … imitates the whole optical system (image transmission)
• 3D-AcquiGen ... simulates the behaviour of CCD camera (image acquisition)

As a result, CytoPacq offers synthetic image data as they would look like if acquired by real microscope and camera. Furthermore, the ideal image of an unaffected specimen is also generated. Hence, the synthetic data can be submitted to any image analysis method and the results of such method can be simply compared to the ideal image and evaluated.

The work was supported by the Czech Ministry of Education (Grants No. 2B06052 and LC535).

References

1. Svoboda D, Kozubek M, Stejskal S. Generation of Digital Phantoms of Cell Nuclei and Simulation of Image Formation in 3D Image Cytometry. Cytometry Part A 2009; 75A: 494–509.

Acquiarium: Free image acquisition and analysis software for image cytometry

Matula P, Daněk O, Maška M, Vinkler M, Kozubek M.

Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Brno, Czech Republic; e-mail: pam@fi.muni.cz

Image cytometry is the measurement of such characteristics as dimension, volume, shape, and reaction kinetics of biological and medical samples. It is usually performed using an optical microscope, scientific camera, and image acquisition and analysis software. Acquiarium (http://cbia.fi.muni.cz/acquiarium) is free software for carrying out the common pipeline of many spatial cell studies using fluorescence microscopy. It can be used for image acquisition and/or image analysis. The image acquisition part is optimized for fast image capture using spinning disk microscopes, which are suitable for 2D or 3D imaging of both fixed and living cells. The image analysis part is focused on comfortable work with a collection of many 3D images. It has a modular design and is extensible via plug-ins. Acquiarium can execute a batch of plug-ins on selected images, which makes extensive analyses possible. Data visualization capabilities of Acquiarium are illustrated in Figure 1.

Image analysis in Acquiarium usually consists of the following steps:

• Raw image correction – It may, for example, include uneven illumination correction, chromatic aberration correction, or alignment of live cell images.
• Object detection in input images (segmentation) – Plug-ins on the basis of thresholding, energy minimization, or mathematical morphology algorithms are implemented. Software builds ‘is-a-part-of’ structure of objects (e.g, nucleoli ‘is-a-part-of’ nucleus). Therefore mutual spatial relations between objects can be studied.
• Object measurement, classification and filtering – Objects can be filtered or classified by computed features (e.g, average intensity, volume, surface area, roundness, location, the number of child objects). Object parameters can be plotted in graphs (histogram or scatter diagrams). Images containing objects with interesting parameters can be found in the collection and accessed in the software by clicking with a mouse in a graph.
• Statistics and summaries – Statistics plug-ins can generate different types of text files summarizing object parameters or compute object statistics.

Acquiarium is intended for the following main applications:

• Quantification of objects, especially dot counting in 2D, 3D, as well as time lapse images and counting the number of larger domains in cells, e.g, the number of nucleoli or protein sites in cell nucleus.
• Spatial arrangement studies. For example, colocalization studies, or radial distribution of hybridization dots in nuclei.
• Measurement of geometrical and shape parameters of cellular structures. For example, volume, surface area, roundness, etc.

The Acquiarium software contains many general purpose algorithms suitable for various image cytometry applications.

We thank all former, current and future developers and collaborators. Grant sponsor: Ministry of Education, Youth and Sports of the Czech Republic (project No. 2B06052).

References

1. Matula P, Maška M, Daněk O, Matula P, Kozubek M. Acquiarium: Free software for the acquisition and analysis of 3D images of cells in fluorescence microscopy. In: IEEE International Symposium on Biomedical Imaging, 4 p, Boston, 2009.

Pozn.: Za věcný obsah abstrakt odpovídají autoři.