Mezinárodní konference Analytical Cytometry V - pokračování

LECTURES

CD4+CD25+ T cells in systemic lupus erythematosus

Blažíčková S.^1,2, Rauová Ľ.^1,3

¹National Institute of Rheumatic diseases, Piestany, Slovak Republic; e-mail: blazickova@laboratoria.sk

²Laboratories Piestany, Slovak Republic

³Department of Pediatrics, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a wide spectrum of clinical manifestations, the loss of tolerance to self-antigens and the production of a vast spectrum of autoantibodies. Recent studies have suggested that CD4⁺CD25⁺ regulatory T (Treg) cells exhibit immune suppressive activity and also play a critical role in the maintenance of self-tolerance. Several studies have shown a significantly decreased percentage of CD4⁺CD25⁺ T-cells in patients with SLE, which correlated inversely with disease activity and/or production of autoantibodies. The decreased frequency of CD4⁺CD25⁺ T cells in adult patients with SLE implies that a deficiency of Tregs is associated with SLE pathogenesis.

In our present study, we examined the frequency of CD4⁺CD25⁺ T-cells in 50 patients with SLE over fourteen years. 37 patients were newly diagnosed before starting any immunosuppresive therapy. The clinical course and activity of the disease, treatment efficacy and level of autoantibodies (dsDNA, DNP, ENA) were followed. The frequency of CD4⁺CD25^+, CD4⁺CD25^+high in whole peripheral blood was analyzed by flow cytometry. We found a decreased of number of CD4⁺CD25^+high T-cells in the group of untreated active patients, which after treatment with a pulse therapy of methylprednisolone was increased. Treatment with cyclophosphamide also restored the decreased number of CD4⁺CD25^high T regulatory cells. There was no correlation between the number of CD4⁺CD25⁺ T-cells and the activity of disease or organ involvement.

Deficiency of Treg may not be the only abnormality of immune regulation in SLE, as combined defects in immune regulation may be responsible for dysregulation of peripheral tolerance and the induction of autoreactivity against self-structure in SLE. The detection and isolation of CD25⁺ Tregs in humans is complicated by the fact that CD25 is a marker of recent T-cell activation. Human peripheral blood contains a heterogeneous compartment of CD4⁺CD25⁺ T-cells, including Treg cells and a considerable amount of active effector T-cells. Nevertheless, we suggest that the decreased number of CD4⁺ CD25⁺ T cells in the peripheral blood of patients with SLE constitutes a defective Treg population and contributes to the pathogenesis of SLE. Our results argue that a breakdown of tolerance as a result of impaired Treg function contributes to the excessive autoantibody formation seen in active SLE.

Which cells are producers of IFN-γ in QauntiFERON-TB Gold in-Tube assay?

Bobovcak M.

Highly Specialized Institute of Lung Diseases, Nová Polianka – Vysoké Tatry, Slovak Republic; e-mail: bobovcak@npolianka.sk

Tuberculosis is the single leading cause of death from any single infectious agent. About one-third of the world population is latently infected with Mycobacterium tuberculosis. Over 8 million new cases and nearly 3 million deaths occur each year. The situation is deteriorating due, in part, to the human immunodeficiency virus pandemic and shifts in the age distribution of the population. Latent tuberculosis infection (LTBI), persists in some, who might develop tuberculosis disease month or years later. The main purpose of diagnosing LTBI is to consider medical treatment for preventing active disease. Interferon-gamma (IFN-γ) release assays (IGRAs) provide a new tool for LTBI diagnosis and surveillance for new TB infection. Two commercial IGRAs are now available: the QuantiFERON-TB Gold In-Tube^® (QFT) assay (Cellestis Limited, Australia) and the T SPOT.TB assay (Oxford Immunotec, UK). IGRAs have features that are advantageous compared with tuberculin skin test (TST) for serial testing. According to manufacturers – they are highly specific and are therefore unaffected by prior bacille Calmette-Guérin (BCG) vaccination; as they can be repeated without concern with boosting effect.

Proclaimed principle of the assays is releasing IFN-γ by specific T effectors upon activation by mycobacterium TB specific antigens (ESAT6, CFP10, TB7.7) in short term in vitro cultivation. Although IGRAs have been recommended for serial testing, data are often confused at the interpretation of new or repeated IGRA results. The data are also often indeterminable. Some existing studies suggest that conversions, reversions and nonspecific variations occur with IGRA serial testing, just as they do with TST serial testing. Published studies also reported inconsistent data for dormant infection in compare with active disease.

We made decision to verify immunological dogma that T cell effectors are the main peripheral blood subpopulation which is activated upon challenging by TB antigens (in short term incubation), used in IGRAs assays. However IGRAs assays tell us nothing about the nature of immunological response in the test tube.

The aim of our preliminary study was phenotypization and enumeration of peripheral blood lymphocytes, which are secreting IFN-γ to culture in QFT Gold whole blood in tube assay.

Heparinised blood of QFT – IFN-γ – ELISA positive patients were retested after new venipuncture with a new set of the testing tubes (NIL control, TB antigen tube and Mitogen PHA tube). After initial 19 hours of incubation, Brefeldin-A was added to all tubes for last 4 hours of cultivation. Immediately after cultivation each sample was stained by antibodies coctail for surface marker staining and IFN-γ antibody for intracellular detection of the cytokine after the following permeabilisation.Cells were analyzed by multicolor multiparameter flow cytometry by the acquisition protocol: ic IFN-γ Fitc /s CD16+56 PE /s CD45 ECD/CD3 PC5/s CD4 PC 7, in CXP software in FC500 Beckman Coulter cytometer. Activation of cells (CD69+ positivity) was determined by another protocol: CD69 PE/CD3 ECD/CD4PC5.

Our study is still running but preliminary data are very interesting. In principle, effectors which could produce IFN-γ in whole blood are subpopulations of CD3+4+ Th1 cells, CD3+8+ Tc cells, NK cells CD3-16+56+ and mixed population of NKT (or NKT like) cells CD3-4± 8± 56+. According to our results: activated population CD3+CD69+ in comparison to CD3-CD69+ population was more included in mitogen PHA stimulation than in TB antigens tube. The CD3-CD69+ cells were prominent population here. However not each activated CD69+ lymfocyte also has produced IFN-γ cytokine.

There are really a few cells in the peripheral blood that are producers of this cytokines upon challenge. In NIL-control tubes was only 1/100 % of IFN-γ+ cells in lymfocytes in average. In TB antigens tubes was approximately from 1/10 to 1 % of IFN-γ+ cells in lymfocytes. QFT Elisa negative patients had the count of these cells in the levels near the detection limit.

The surprise of the study is the result of phenotypisation of IFN-γ+ lymfocytes. While in PHA stimulation (PHA is strong T-cell activator) Th and Tc population was dominated (CD3+4+56-, CD3+4-56-), in TB antigens stimulated samples the IFN-γ+ NKT (or NKT like, CD3+4± 56+) and NK (CD3-4-56+) cells were prevailing subpopulation. We also reported only the minority of Th and Tc cells which responded to these antigens. The interesting observation resulted from dividing of QFT positive subjects according to diagnoses: old cured nonactive TB, active TB and LTBI. There was also identified T cell component of the response in old TB and active TB, which almost missed in latent TB patients.

While NK cells could be secondarily stimulated by released IFN-γ paracrine and autocrine way (and by others cytokines like IL1), NKT cells could be stimulated by specific stimulus via TCR receptor. TCR receptor of NKT can bind not only classic lipid antigens but also larger peptides with hydrophobic properties. Mycobacterial peptides ESAT6 and CFP10 used in IGRA assay are such type of antigens. NKT cells represent the link between native and acquired immune response and have regulatory, effector and also memory properties. NKT cell is strong and rapid producer of IFN-γ in some situations. Their role in TB pathogenesis and granuloma formation in vivo is not totally uncovered. We have observed also CD3 (TCR) downregulated population of cells in TB antigens tubes (CD3-4±56-, CD3-4+56+). This situation is often observed upon T cell activation. There could be also masked TB antigen activated T-helper and T-cytotoxic clones (and probably also NKT cells) inside this downregulated population.

Our observations offer some new important problems to solve. Which population reacts earlier and which are secondary responsed: T, NKT or NK ? In future we are going to gather data from more patients and answer also these kinetic questions. The important question is also to determine phenotypes of these NKT - IFN-γ producers. Peripheral NKT or NKT like cells are functionally and phenotypically mixed population w/wo restringed repertoire of TCR, etc. These studies could answer the problems with interpretation or misinterpretation of such type TB tests. It could be also helpful for novel assays designing.

Regulatory T cells in patients with head and neck squamous cell carcinoma

Boucek J.¹, Mrkvan T.², Chovanec M.¹, Kuchar M.¹, Betka J.¹, Boucek V.⁴, Hladikova M.⁴, Betka J.¹, Rihova B.², Eckschlager T.³

¹Charles University, 1^st Faculty of Medicine, Dept. Otorhinolaryngology Head and Neck Surgery, University Hospital Motol, Prague, Czech Republic

²Institute of Microbiology AS CR, Prague, Czech Republic

³Dept. Paediatric Haematology Oncology and Dept. Medical Informatics, 2^nd Faculty of Medicine, Charles University, Prague, Czech Republic; e-mail: eckschlagertomas@yahoo.com

⁴Dept. Haematology Blood transfusion, Hospital Rudolf and Stephania Benesov, Czech Republic

Regulation of immune responses plays an important role in the maintenance of homeostasis including suppression of cancer. Immune system is indeed complex and highly regulated to suit the purpose of ensuring responses to dangerous substances or organisms, and tolerating those which are harmless for the host. Several mechanisms are employed to safeguard those substances which are considered harmless, including self-antigens that do not cause any overt activations of the immune system, one of which is peripheral immune tolerance. Although significant advances in the field of treatment of patients with Head and Neck Squamous Cell Carcinoma (HNSCC) have been documented within last years, regrettably, survival rates for this disease have not improved over many years (Forastiere et al., 2001). Thus, the development of new molecular markers which could help to describe the biological and immunological status of the patients and predict the progression of the disease seems to be promising. Relationship between immune system and cancer cells is very important and decisive for the progression of malignancy. Among others, T regulatory cells are a crucial subset which plays a role in the control of tumor growth (Strauss et al, 2007).

Blood samples collected at the time of diagnosis were obtained from 112 HNSCC patients who underwent curative therapy. All patients were examined at the Dept. Otorhinolaryngology and Head and Neck Surgery, Univ. Hosp. Motol. We used 20 peripheral blood samples from healthy volunteers as controls. Peripheral blood samples were analyzed by flow cytometry (FACSCalibur). Used antibodies: anti CD45 FITC/CD14 PE for correct lymphocyte gating, anti CD3 FITC/CD19 PE, anti CD3 FITC/ CD16CD56 PE, anti CD4 FITC/ CD8 PE, anti CD45RA FITC/anti CD4 PE and anti CD3 FITC/ CD4 PE/CD25 APC (Beckmann Coulter). Results were expressed as the percentage of appropriate cells in lymphocytic gate. Total blood count and biochemical and tumor makers were also examined. To study the relation between different categories, the data were classified in a frequency 2 × 2 table χ² test with Danderar’s correction. Numerical data were presented as mean ± standard deviation and were analyzed using Student’s t-test. The correlations between immunological parameters and early recurrence of disease were evaluated by nonparametric Spearman’s coefficient. P values of < 0.05 were considered significant. Software SPSS v. 10.1 was used for statistical calculations.

The percentage of circulating CD3⁺CD4⁺CD25⁺ and CD4⁺ cells was increased while NK cells (CD3^-CD16⁺CD56⁺) were decreased in patients with HNSCC relative to those in controls. Platelets count and α-1-antitrypsin were higher in patients with tumors of hypopharynx than in those with tumors of base of the tongue. (There were no statistically significant differences in T- and N-stages and tumor grading between different tumor localizations). The group of patients was divided according to the size of primary tumor (T1–T4 stage), the spread of tumor to the regional lymphatic nodes (N vs. N⁺) and grade (distant metastases – M1 were not detected in any patient at the time of diagnosis). There was increase of tumor markers SCC; AAT, Cyfra-21, CRP and ratio of naēve T lymphocytes and level of CRP with increasing size of the primary tumor (T). The level of tumor marker Cyfra-21 and CRP were higher and the percentage of B cells was lower in N⁺ group than in N0. We found higher proportion of CD8⁺ and lower CD4⁺/CD8⁺ ratio in patient with less differentiated tumors (G1+G2 vs. G3+G4). The group of patients with recurrent disease was compared to the group without evidence of disease over a follow-up interval longer than one year. The increase of Treg cells and AAT and decrease of erythrocyte count and hemoglobin levels was found in group with early recurrency.

We have observed increased percentage of circulating CD4⁺CD25⁺ T cells in the peripheral blood in patients with HNSCC similarly to reported data. All hitherto published data have shown that the total amount of Treg in peripheral circulation of HNSCC is two-fold higher than in controls, although the exact numbers are slightly different among the different laboratories (Schaefer et al, 2005; Strauss et al, 2007). In an interval of follow-up longer than one year we compared the group of patients with recurrent disease with disease-free group. We found a difference in the level of T regulatory cells at the time of primary diagnosis between patients in remission and in relapse. The level of T regulatory cells in peripheral blood correlates with a higher probability of early recurrence of HNSCC. This finding has helped us to select patients eligible for extensive therapy and more intensive follow-up. One may speculate that erythrocyte count, hemoglobin level and regulatory T cell proportion may be useful as predictive factors in HNSCC.

This work was supported by the Ministry of Education, Youth and Sports of The Czech Republic - Grant MSM 0021620813.

References

1. Forastiere A, Koch W, Trotti A, Sidransky D. Head and neck cancer. N Engl J Med 2001; 345: 1890–1900.

2. Schaefer C, Kim GG, Albers A, Hoermann K, Myers EN, Whiteside TL. Characteristics of CD4+CD25+ regulatory T cells in the peripheral circulation of patients with head and neck cancer. – Br J Cancer 2005; 92: 913–920.

3. Strauss L, Bergmann C, Gooding W, Johnson JT, Whiteside TL. The frequency and suppressor function of CD4+CD25highFoxp3+ T cells in the circulation of patients with squamous cell carcinoma of the head and neck. Clin Cancer Res 2007; 13: 6301–6311.

Regulatory T cells in autoimmune diseases

Kral V., Pohorska J.

Institute of Public Health, Ustí nad Labem, Czech Republic; e-mail: vlastimil.kral@zuusti.cz

The concept of polarization of the immune response was one of the main topics on the field of immunology in the end of 20^th century. There was expectation to solve the problem of diagnostics, therapeutics and control of allergy and autoimmune diseases. Recently we are witnessing analogical situation. Abnormalities in newly discovered T cells subsets – regulatory T cells (Treg) and proinflammatory IL-17 producing helper T cells (Th17) have been implicated in autoimmune, allergic and immunoinflammatory conditions (Afzali, 2007; Oukka, 2007). Many studies, both in mice and humans, have confirmed the importance of T reg subset in the pathogenesis of many autoimmune diseases (i.e. thyroiditis, gastritis, RA, SLE, etc.) (Toubi, 2008)

Treg immunophenotype was discovered as CD4⁺CD25⁺FoxP3⁺. However, in humans the expression of specific transcription factor FoxP3 is not exclusively confined to CD4⁺CD25^hi cells. Naive CD25 ^lo and activated T-cells are able to express FoxP3. It was found that IL-7 receptor (alpha chain of IL-7R, CD127) is down-regulated on human Treg cells and immunophenotype CD4⁺CD25^int-hiFoxP3⁺CD127^lo-dim of human Treg was postulated (Liu, 2006).

Treg immunophenotyping by using both CD127 and FoxP3 markers was done by 4-colour flow cytometry. Following groups of patients and controls were investigated: 1) autoimmune diseases patients (rheumatoid arthritis, RA, n = 36, ulcerative colitis, UC, n = 9, Crohn’s disease, CD, n = 28, psoriasis, PS, n = 26); 2) healthy controls, HC, n = 14; 3) other immunopathology conditions (allergy, AL, n = 15; infection (latent and/or treated TBC), IF, n = 22).

Results of Treg cells are following (values represent the portion of CD4⁺ population): 1) RA 7.4 ± 3,0% (patients on biological therapy, n = 8) and 5.9 ± 2.4% (DMARD’s treated, n = 28); PS 7.6 ± 4.0% (x_min - x_max range 1.8–20,4); UC 4.0 ± 1,4 (x_min 0.7- x_max 5,6); CD 6.7 ± 2,1% (x_min 3.5 - x_max 11.7); 2) HC 5.9±1,1% (x_min 4.0 - x_max 7.5) ; 3) other patients groups: AL 6.6±1.1 (x_min 4.9 - x_max 8.8); IF 8.5±2.9 (x_min 3.8 - x_max 16.6).

There was shown that Treg cells vary (up and down, see PS group for example) in autoimmune diseases in comparison to HC and AL groups. A visible tendency to higher Treg levels was found at IF group (latent and, more profoundly, treated TBC). For achieved results analysis there would be necessary to have the complete data of conventional and/or biological therapy of autoimmune disease patients. However, there is another knowledge probably influencing interpretation of Treg immunophenotyping results – the evidences of impaired function of regulatory T cells when immunophenotype is quite normal (Afzali, 2007).

Treg determination by means of CD127 and FoxP3 provides the quite well reproducible results and this method would be useful for clinical research.

References

1. Afzali B, Lombardi G, Lechler RI, Lord GM. The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clin Exp Immunol 2007; 148: 32–46.

2. Liu W, Putnam AL, Xu-yu Z, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4⁺ T reg cells. – JEM 2006; 7: 1701–1711.

3. Oukka M. Interplay between pathogenic Th17 and regulatory T cells. Ann Rheum Dis 2007; 66: 87–90.

4. Toubi E. Targeting T Regulatory Cells in Autoimmune Diseases. IMAJ 2007; 10: 73–76.

Basophil activation test in food allergy

Sedlackova L., Prucha M.

Department of Clinical Biochemistry, Haematology and Immunology, Hospital Na Homolce, Prague, Czech Republic; e-mail: lenka.sedlackova@homolka.cz

The food allergy is a growing problem to diagnose, as it is to treat. The traditional diagnostic tools – specific IgE and skin prick tests – do not have optimal sensitivity and specificity. The gold standard of diagnostic – double blind placebo controlled exposition test – is not widely applicable in routine praxis. The functional laboratory tests such as basophil activation test bring new hope to solve this situation. They are easy to perform and in contrast to clinical tests are completely save for patients.

The aim of the study was to establish basophil activation test in routine immunology laboratory diagnostics of food allergy, to evaluate performance characteristics of this test and to compare its results with specific IgE.

Children and adult patients with symptoms suspected from food allergy were tested for basophil activation test and specific IgE to milk, egg, wheat flour, soybean, peanut and walnut.

Data will be presented including comparison of both methods. A small proportion of patients are non-responders and it is not possible to evaluate their basophil activation test. Despite of this our first results are very promising and the basophil activation test is useful tool in food allergy diagnostics.

Tissue specific stem cell markers used in cytometry

Klíma J., Hlučilová J., Juhás Š., Vodička P., Motlík J.

Institute of Animal Physiology and Genetics v.v.i., Academy of Sciences of Czech Republic, Liběchov; e-mail: klima@iapg.cas.cz

Stem cells represent a privileged cell population possessing self renewal capability and having potential to differentiate into various cell types. Researchers can exploit only few putative stem markers or particular properties in which they differ from other somatic cells. There are several strategies to identify and isolate them. The most crucial and most commonly approach uses immunophenotyping of stem population mostly evaluated via flow cytometry (Challen et al., 2009) or histochemistry. Moreover a majority of surface or even intracellular markers are not directly related to determination or maintenance of stemness. An alternative method is based on side population (SP) detection that was successful in many adult tissues (Challen et al., 2009; Kim and Morshead, 2003; Redvers et al., 2006). SP cells having stem or progenitor properties export xenobiotics in non-specific manner and could be thus identified by flow cytometry. Aldehyde dehydrogenase activity is another example of stem cell marker that is not involved in stemness determination but is coexpressed (Levi et al., 2009). More rarely used marker for mainly quiescent stem cells is the small cell size that could be determined via various methods based on image analysis or electronic volume determination.

A brief overview on limitations of each approaches and possibilities how to combine those for the purpose of better identification of stem cells will be given. Selected markers and methods will be presented in several studies on neural, epidermal and bone-marrow derived mesenchymal stem cells of mammals.

This work was supported by grants 2B06130, 1M0538 and AV0Z50450515.

References

1. Challen GA, Boles N, Lin KK, Goodell MA. Mouse hematopoietic stem cell identification and analysis. Cytometry A 2009; 75: 14–24.

2. Kim M, Morshead CM. Distinct populations of forebrain neural stem and progenitor cells can be isolated using side-population analysis. J Neurosci 2003; 23: 10703–10709.

3. Levi BP, Yilmaz OH, Duester G, Morrison SJ. Aldehyde dehydrogenase 1a1 is dispensable for stem cell function in the mouse hematopoietic and nervous systems. Blood 2009; 113: 1670–1680.

4. Redvers RP, Li A, Kaur P. Side population in adult murine epidermis exhibits phenotypic and functional characteristics of keratinocyte stem cells. Proc Natl Acad Sci USA 2006; 103: 13168–13173.

Expression of neuronal markers during fetal and postnatal period in the miniature pig spinal cord

Hlucilova J.¹, Cizkova D.², Juhas S.¹, Strnadel J.¹, Motlik J.¹, Marsala M.³

¹Institute of Animal Physiology and Genetics, AS CR, v.v.i., Liběchov; e-mail: hlucilova@iapg.cas.cz

²Institute of Neurobiology, Slovak Academy of Sciences, CE, Košice

³Anesthesiology Research Lab, University of California, San Diego

Doublecortin (DCX) is a microtubule-associated protein expressed in migrating and differentiating neuronal precursors during embryonic development of CNS and within well-marked areas of adult brain (Couillard-Despres et al., 2005). Neuronal precursors begin to express DCX shortly after exiting the cell cycle, and continuing to express DCX for 2-3 weeks as the cells mature into functional neurons. Afterwards a mature neuronal marker NeuN (Neuron-specific nuclear protein) starts to be detectable. Therefore, DCX seems to be a suitable marker for neurogenesis in developing organisms or for determining neurogenic regions in adulthood (Brown et al., 2003). Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is expressed in astrocytes and ependymal cells in CNS. Substantive evidence now indicates that adult NSCs express GFAP and exhibit properties associated with glia both in vivo and in vitro. These findings raise important questions regarding the relationship of GFAP-expressing neural stem cells to GFAP-expressing astrocytes (Zhu and Dahlström, 2007). Moreover GFAP decreases have been reported in Down’s syndrome, schizophrenia, bipolar disorder and depression.

The aim of this study was to observe expression of DCX, NeuN and GFAP in the miniature pig spinal cord in four fetal stages (40, 60, 80 and 100 d), two postnatal periods (newborn and 14 d) and in adult pig. Frozen spinal cord sections were taken from cervical, thoracic and lumbar spinal cord. Free-floating sections were double- immunoassayed with a combination of DCX (goat anti-Doublecortin, Santa Cruz Biotechnology) and NeuN (Mouse anti-Neuronal Nuclei, Chemicon) antibodies or double – immunoassayed with a combination of DCX (goat anti-Doublecortin, Santa Cruz Biotechnology) and GFAP (Mouse anti-GFAP, Sigma) antibodies. Hippocampus from adult rat was used as a positive control for DCX expression in neuronal precursors. Moreover we isolated, cultivated and differentiated spinal cord neural stem cells in vitro. These cells were harvested from 80 days gravidity fetuses. The neurospheres were dissociated at day 7 by mechanical trituration and re-plated and processed either for proliferation or for differentiation studies. After differentiation (7, 14, 21 days) the cells were fixed and incubated with primary antibodies for developmental neuronal markers DCX (goat-polyclonal IgG antibody 1 : 200 (Santa Cruze, CA), Tuji (mouse IgG antibody 1 : 1000, ßIII-tubulin, Chemicon), MAP2 (rabbit IgG antibody 1:1000, Chemicon).

Staining of spinal cord sections with DCX antibodies revealed strong positivity in 40 days old fetus, immune-reactivity was detected in all neurons and neuropils. This fetal stage was connected with no GFAP presence in whole spinal cord. In 60 and 80 days old fetuses we observed weaker expression of DCX (maximum expression in dorsal horn, ventral horn and central canal) but there were great number of migrating DCX positive cells and also number of NeuN positive cells was rapidly increased. In 60 days old fetuses the GFAP spinal cord expression started. GFAP positive cells were firstly located in the periphery of dorsal, ventral and lateral funiculus as well as in the surroundings of central canal. In 80 days old fetuses the GFAP expression expanded into white matter. The 100 days old fetuses showed only lightly DCX positivity in dorsal horn. On the other side GFAP positivity was present in whole white matter. In newborn pig, doublecortin antibody we only detected in dorsal horn (lamina II and III) of lumbar spinal cord. In addition GFAP expression was slightly observed in gray matter of spinal cord. No DCX immunoreactivity we detected in 14 days old miniature pig as well as in adult porcine spinal cord. Moreover GFAP positivity in white and grey matter of adult miniature pigs was increased in comparison with spinal cords of newborn piglets. Neuronal differentiation of neural progenitors isolated from porcine fetal spinal cord (F80) under in vitro conditions at 4–21 days showed that bipolar, primitive neuronal precursors expressing DCX could be seen already at 4 days. Multipolar ramified DCX positive neuronal progenitors occurred at 7–10 day. Further differentiation showed only immature TUJI and MAP2 positive fully differentiated neurons.

This data demonstrate that DCX was expressed in early post-mitotic spinal cord neurons during embryonic and fetal stage, but was absent in the adult naive spinal cord in miniature pig. This result we also confirmed by Western blot analysis. Marked decrease of DCX positivity in spinal cord fetuses was occurred in the third trimester of pregnancy. NeuN and GFAP immunoreactivity was increased with progressive embryonic development. Miniature pig neural stem cells isolated from spinal cord were able differentiate into neurons in vitro.

This work was supported by AV0Z50450515, 1M0538, APVV-51002105, APVV SK-CZ 0045-07, and MEB0808108, 2B06130.

References

1. Brown JP, Couillard-Després S, Cooper-Kuhn CM., Winkler J, Aigner L, Kuhn HG. Transient expression of doublecortin during adult neurogenesis. J Comp Neurol 2003; 467: 1–10.

2. Couillard-Despres S, Winner B, Schaubeck S, Aigner R, Vroemen M, Weidner N, Bogdahn U, Winkler J, Kuhn HG, Aigner L. Doublecortin expression levels in adult brain reflect neurogenesis. Eur J Neurosci 2005; 21: 1–14.

3. Zhu H, Dahlström A. Glial fibrillary acidic protein-expressing cells in the neurogenic regions in normal and injured adult brains. J Neurosci Res 2007; 85: 2783–2792.

Osteogenic differentiation of miniature pig mesenchymal stem cells in 2D and 3D environment

Juhas S., Hlucilova J., Klima J., Strnadel J., Holubova M., Motlik J.

Institute of Animal Physiology and Genetics, AS CR, v.v.i., Liběchov; e-mail: juhas@iapg.cas.cz

Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into a variety of cell types (osteoblasts, chondrocytes, myocytes and adipocytes). They are also the primary cell source for cartilage and bone formation and so may be optimal for cell-seeded scaffolds and can be used for bone tissue repair (Sumanasinghe et al., 2006). Several studies reported that porcine MSCs are a valuable model system for mesenchymal basic research and tissue engineering (Ringe et al., 2002).

Therefore in this study we studied miniature pig MSCs osteogenic ability by various evaluation methods in different cultivation conditions and systems. The porcine MSCs cultured in the growth medium up to the 3^rd passage(CD29⁺, CD44⁺, CD90⁺, CD105⁺ and CD45^-), were transferred into Petri dishes (2D environment) un/coated with vitronectin and collagen I or embedded in plasma clot (PC) scaffolds (3D environment). Afterwards these cells were cultured in osteogenic medium containing glycerol 2-phosphate, ascorbic acid 2-phosphate and dexamethasone. During three weeks of differentiation, the formation of nodules and deposition of calcium were visualized by Alizarin red S staining. Expression of osteopontin, osteocalcin and osteonectin as osteogenic markers was evaluated by immunocytochemistry and Western blot analysis. We also detected the alkaline phosphatase in 2D system.

Osteocalcin was started to be observed in 2D environment on 7^th day of osteo-differentiation. On the other side in PC scaffolds osteocalcin expression was immunostained on 14^th day at first. After one week of 2D and 3D differentiation osteonectin was determined and its expression persisted to the final collection. We already detected high expression of osteopontin at the creation of PC scaffolds and 100% confluence of MSCs in control medium. Osteopontin was presented during osteogenic differentiation and also control cultivation but the expression signal was more intensive in osteogenic medium. After 7 days of differentiation in multilayers and after 21 days of differentiation in PC scaffolds we observed decrease of osteopontin visualization. Moreover we detected the osteonectin expression during whole osteo-cultivation with maximum on the 7^th (ECM coated dishes) or 14^th (ECM uncoated dishes) day of differentiation by Western Blot analysis. There is evidence that contact with vitronectin and collagen I promotes the osteogenic differentiation of human MSCs, and that ECM contact may be sufficient to induce differentiation in these cells (Salasznyk et al., 2004). Therefore we used this methodology and we found out specific differences in osteogenic markers expression in MSCs differentiated on uncoated and coated dishes. At the beginning of the differentiation (100% confluence of cells) there was approximately two-fold increase of osteonectin and osteopontin expression in MSCs growing on ECM coated dishes in comparison with uncoated dishes. This result was in accordance with above-mentioned study.

We suggest that MSCs seeded on ECM coated dishes started already differentiate without osteogenic medium during proliferation in control medium before gaining of the total confluence. Time path of osteopontin expression in differentiated MSCs with or without ECM molecules also support results concerning osteonectin detection. Our study indicates comparable ability of miniature pig MSCs osteo-differentiation in 2D and 3D environment.

This work was supported by AV0Z50450515, 1M0538, and 2B06130.

References

1. Ringe J, Kaps C, Schmitt B, Büscher K, Bartel J, Smolian H, Schultz O, Burmester GR, Häupl T, Sittinger, M. Porcine mesenchymal stem cells. Induction of distinct mesenchymal cell lineages. Cell Tissue Res 2002; 307: 321–327.

2. Salasznyk RM, Williams WA, Boskey A, Batorsky A, Plopper GE. Adhesion to Vitronectin and Collagen I Promotes Osteogenic Differentiation of Human Mesenchymal Stem Cells. 2004; 1: 24–34.

3. Sumanasinghe RD, Bernacki SH, Loboa EG. Osteogenic differentiation of human mesenchymal stem cells in collagen matrices: effect of uniaxial cyclic tensile strain on bone morphogenetic protein (BMP-2) mRNA expression. Tissue Eng 2006; 12: 3459–3465.

Irradiation of dental pulp stem cells and periodontal ligament stem cells via ⁶⁰Co – apoptosis or senescence?

Visek B.¹, Soukup T.¹, Rezecova M.², Muthna D.², Bruckova L.³, Kucerova L.⁴, Jiroutova A.², Suchanek J.⁵, Mokry J.¹

¹Department of Histology and Embryology, Charles University in Prague, Medical Faculty in Hradec Králové; e-mail: viseb6ar@lfhk.cuni.cz

²Department of Medical Biochemistry, Charles University in Prague, Medical Faculty in Hradec Králové

³Faculty of Chemical Technology, University of Pardubice

⁴Department of Clinical Genetic, Teaching Hospital in Hradec Králové

⁵Department of Stomatology, Teaching Hospital in Hradec Králové

Dental pulp stem cells (DPSC) and periodontal stem cells (PLSC) are elements which significantly participate in regenerative processes in oral cavity. Their main role is maintenance of tissue homeostasis and regeneration of damaged tissues. More, many patients nowadays undergo radiotherapeutic treatment that is focused on oral tissues. So we put a question – what is the most expressive reaction of DPSC and PLSC to the irradiation? Can these cells hold their proliferation kinetic parameters or not? Which changes in cell cycle can we observe? These and some other questions are the particular aims of our work.

In this work we used gamma rays of ⁶⁰Co for irradiation. Applied doses were 2, 6 and 20 Gy. Moreover, we compared the irradiated cell lineages with control untreated cells. To the experiment were used 3 cell lineages (2 × DPSC, 1 × PLSC). These cell lineages were cultivated in MAPC media, contained PDGF + EGF both before and after irradiation. Cells were analyzed in days 1, 3, 6 and 13 after irradiation. Viability and proliferation activity were analyzed in ViCell XR and Z2 Counter (both Beckman Coulter), respectively. Cell cycle was assessed by using of 2 methods, both staining with propidium iodide and dual staining with anti-cyclin A2/7-AAD. Early and late phase of apoptosis were detected via Annexin V/7-AAD and analyzed by using flow cytometer Cell Lab Quanta SC+MPL (Beckman Coulter).

Both in light and phase microscope, irradiated cells did not show significant morphological changes. Cumulative number of population doublings was decreased both in DPCS and PLSC (DPSC: 5.8 (control lineage), 2.7 (2 Gy), 1.0 (6Gy) and 0.9 (20 Gy); PLSC: 5.3 (control lineage) and 1.4 (20Gy)). Viability showed decrement from 92.0% (control lineage) to 88.2% (2 Gy), 87.1% (6Gy) and 87.0% (20 Gy) in DPSC. Viability in PLSC decreased from 95.0% (control lineage) to 90.9% (20 Gy). In cell cycle phases we observed increased number of G2/M-phase in irradiated cells depending on the used dose. Percentage share of G2/M phase in DPSC was 12%, 13% and 63% in control lineage, 2 Gy and 20 Gy, respectively. PLSC showed increment from 11% to 45% in control lineage and 20 Gy, respectively. All the irradiated lineages showed high expression of beta-galactosidase. Both markers of early (Annexin V+/7-AAD-) and late (Annexin V+/7-AAD+) apoptosis in irradiated cells were not significantly increased compare to the control lineages. In karyotype we observed changes in all the irradiated lineages depend on the used radiation dose.

We concluded that the main reaction of the DPSC and PLSC to the irradiation is the senescence. There was observed the block in G2 phase of cell cycle and proliferation activity was significantly decreased, depend on used dose. There was not observed signs of apoptosis in all the cultivated and irradiated cells. The minimal changes of monitored parameters we observed in 2 Gy irradiated cell (both DPSC and PLSC). So we suppose that this dose is potentially acceptable in clinical applications.

This work was supported by grant GA CR 304/09/1568.

External quality control of CD34⁺ stem cell enumeration: a multicenter analysis

Lysák D.¹, Kalina T.², Martínek J.³, Pikalová Z.⁴, Vokurková D.⁵, Jarešová M.⁶, Marinov I.⁷, Ondrejková A.⁸, Špaček M.⁹, Stehlíková O.¹⁰

¹University Hospital Plzeň, Plzeň, Czech Republic; e-mail: lysak@fnplzen.cz 

²Motol University Hospital, Prague, Czech Republic

³P&R Lab a.s., Nový Jičín, Czech Republic;

⁴University Hospital Olomouc, Olomouc, Czech Republic

⁵University Hospital Hradec Králové, Hradec Králové, Czech Republic

⁶Institute for Clinical and Experimental Medicine, Prague, Czech Republic

⁷Institute of Hematology and Blood Transfusion, Prague, Czech Republic

⁸Krajská nemocnice T. Bati, a.s., Zlín, Czech Republic

⁹University Hospital Královské Vinohrady, Prague, Czech Republic

¹⁰The Faculty Hospital Brno, Brno, Czech Republic

CD34⁺ cell enumeration is one of the basic laboratory methods in haemato- oncology. Many clinical decisions are made on the basis of the results of this measurement (stem cell collection timing, graft quality assessment etc.). Therefore this method is expected to be adequately accurate and precise. The intra- and inter- laboratory variability should be minimal.

We performed a multicenter measurement of CD34⁺ cells. The aim of the project was to assess the inter-laboratory variability and verify the performance of participating laboratories. Since 11/2007 three send-outs were organized. Stabilized blood samples were sent into 10 Czech flow cytometry laboratories and analyzed according to the local protocols. WBC and CD34⁺ counts and basic methodological details were reported and statistically evaluated. A special scoring system was created comparing the measured value to the distribution of values reported by all laboratories (correct values have score -1 to +1). Most laboratories (9/10) have used ISHAGE based gating protocols (one laboratory Milan protocol) and double platform method.

Between-laboratory variability for CD34+ percent and CD34+ count (cells/Ķl) was higher during 1^st control cycle (43 % and 58 %) in comparison to the later cycles (cycle 2.: 30 % and 32 %; cycle 3.: 29 % and 31 %). Following the 1^st send-out the average score per sample has declined for CD34+ % (4.79 → 1.96 → 1.28) as well as for CD34+/μl (2.04 → 2.41 – new lab joined the project → 1.16).

The project was effective in reducing between-laboratory variability and resulted in more comparable results reported by participating laboratories. The control cycles also appeared to be useful as a tool of proficiency testing of the laboratory technicians. The analysis confirmed that the participation of the flow laboratory in some between-laboratory measurement or the external quality control scheme is an important part of the good laboratory practice and quality management system.

The study is supported by the grant of the Ministry of Health of the Czech Republic (IGA NR/ 9268-3).

Technical aspects and guidelines for application of EuroFlow protocols: towards 8-color flow cytometry in the diagnosis and classification of hematopoietic malignancies

Kalina T.¹, Flores-Montero J.², Lécrevisse Q.², Cullen M.³, Lhermitte L.⁴, Sedek L.⁵, Mendonća A.⁶, Böttcher S.⁷, te Marvelde J.⁸, Mejstrikova E.¹, Hrusak O.¹, van Dongen J. J. M.⁸, Orfao A.² – On behalf of the EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708)

¹Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic; e-mail: tomas.kalina@lfmotol.cuni.cz

²Department of Medicine, Cancer Research Centre (IBMCC-CSIC-USAL) and Cytometry Service, University of Salamanca, Salamanca, ES

³St. James University Hospital, Leeds, UK

⁴Department of Hematology, Hôpital Necker, Paris, FR

⁵Department of Pediatric Hematogy and Oncology, Medical University of Silesia, Zabrze, PL

⁶Department of Hematology, Instituto Portugues de Oncologia, Lisbon, PT

⁷2^nd Department of Medicine, University Klinik Schleswig-Holstein, Kiel, DE

⁸Department of Immunology, Erasmus MC, Rotterdam, NL

EuroFlow collaborative project is aimed at designing approaches for fast and sensitive diagnostics of hematopoietic malignancies using 8-color flow cytometry. We have extensively tested approaches for instrument settings, compensation, choice of fluorochromes and staining protocols. We have developed new analysis software “Infinicyt” for advanced semi-automated analysis of 8-color flow cytometry data.

When a test cohort of 30 healthy donors’ peripheral blood was measured using the common Euroflow protocol in 8 participating laboratories, we could demonstrate the coefficients of variation of each parameter to be lower then 50%. Furthemore, we could merge the data files into one file that is amenable to analysis in the Infinicyt software where all expected cell types cluster to the same position (Fig. 1). In conclusion, we show that 8-color flow cytometry is useful tool for diagnosis and classification of hematopoietic malignancies.

Cytometry reveals differentiation in hematopoiesis

Hrušák O., Mejstříková E., Kalina T.

CLIP-Cytometry, Dept. of Pediatric Hematology and Oncology, Charles University and Univ. Hospital Motol, Prague; e-mail: Ondrej.Hrusak@lfmotol.cuni.cz

Clinical hematologists consider flow cytometry as the most useful laboratory investigation in acute leukemia (AL) diagnostics, as demonstrated in a recent poll. This is certainly influenced by a very good accessibility of diagnostic material – most importantly, bone marrow. Very good collaboration of clinical centers of the Czech Pediatric Hematology Working Group with our reference laboratory enables a population-based analysis. This shows the incidence of cell lineages (and cells at various stages of differentiation) that may undergo leukemic transformation. Subtype definition (based on the criteria of the European Group for Immunophenotyping of Leukemias, EGIL) is simple. However, there is a striking inconsistency worldwide in the way how definition is interpreted in everyday life. Therefore, there is a lack of consecutive cohorts of patients, which would show efficacy of different types of chemotherapy in borderline (=hybrid) AL. In our cohort of 694 pediatric patients, treated from 1996 to 2006 we show that the 5-year event-free survival is 75 ± 2.1% and 47 ± 4.9%, following the lymphoid- or myeloid-directed therapy. In addition, we show various definitions of hybrid AL (AHL). Based on the uniform criteria, most of our AHL patients receive a lymphoid-directed therapy. Although the prognosis of AHL is generally poorer than that of non-hybrid ALL, we found no indication for a general use of myeloid-directed treatment in these patients. We show a usefulness of cytometric techniques in combination with molecular genetics and novel statistical approaches in the definition of AHL and other borderline AL subtypes.

Supported by IGA NR/9531-3, NPV 2B06064 and MŠMT MSM0021620813.

Dynamics of malignant and non malignant cells in bone marrow during the early phase therapy of childhood ALL; correlation with genetic changes using polychromatic FACSorting

Mejstříková E.¹, Froňková E.¹, Lhermitte L.², Semerák P.¹, Kováč M.¹, Kalina T.¹, Hrušák O.¹

¹CLIP-Cytometry, Dept. of Pediatric Hematology and Oncology, Charles University and Univ. Hospital Motol, Prague; e-mail: ester.mejstrikova@lfmotol.cuni.cz

²Department of Hematology, Hôpital Necker, Paris, France

Bone marrow represents dynamic tissue with a complex cellular composition. Origin of leukemia interpheres with non malignant hematopoiesis just not only on the basis of mechanical suppression but very probably also by soluble factors produced by non malignant and malignant cells. Therapy of leukemia represents a specific situation where reduction of blasts is accompanied by the reappearance of normal hematopoiesis. Leukemic cells non-randomly change its phenotype.

In patients with de novo ALL and in whom we evaluated MRD and reconstitution of non malignant hematopoiesis where analyzed in software FlowJo (4-color data, 10/2002 – 10/2007) and Infinicyte (8-color data, data since 11/2007 till now). WBC counts with differential count and hierarchical clustering analysis of initial immunophenotypic data were analyzed in recruited patients.

Background identified by 4-color was high at later time points when recovery of hematopoiesis is typical (day 33 and week 12) and this background hampered sensitivity in these time points. 8-color FC leads to more fine separation of non malignant and malignant cells. We identified a novel subgroup of patients in who significant instability of immunophenotype leads to monocytic phenotype during the treatment.

To conclude, 8-color FC represents perspective methodology not only for MRD but also for the identification of novel ALL subtypes.

Supported by IGA NR/9531-3, NPV 2B06064 a MZ CR 000064203, MSM0021620813.

Detection of minimal residual disease of chronic lymphocytic leukemia by flow cytometry using the Rawstronęs protocol – a reliable tool for clinical evaluation of CLL therapy

Stehlíková O., Hrabčáková V., Francová H., Brychtová Y., Doubek M., Mayer J., Klabusay M.

Laboratory of Flow Cytometry and Cellular Therapy, Centre of Molecular Biology and Gene Therapy, Dept. of Internal Medicine – Hematooncology, University Hospital, Brno; e-mail: vhrabcakova@fnbrno.cz

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adult population. Prognosis of the disease is evaluated with respect to clinical as well as molecular parameters. After the modern intensive therapy regimens using monoclonal antibodies or allogeneic transplantation of hematopoietic stem cells, complete remission (CR) of the disease is often reached, according to the NCI-WG criteria. However, in some patients, leukemic cells are still present at low counts (often less than 1of 10⁴ of leucocytes) as minimal residual disease (MRD). If present, these cells can cause a relapse of the disease after the treatment was ended. Rawstron et al. (Leukemia, 2007) developed an international standardized 4-color flow cytometry protocol on minimal residual disease detection and the data were also matched with molecular evaluation of MRD level. Due to specific phenotype of CLL or SLL (small-cell lymphocytic lymphoma) tumor cells, it is possible to determine the MRD level in peripheral blood or bone marrow down to less than 0.01% of leucocytes.

We used the flow cytometry protocol for MRD detection in routine monitoring of ten CLL patients after allogenic stem cell transplantation. Four basic flow cytometry protocols using a combination of 4 monoclonal antibodies (anti-CD3, 5, 19, 20, 22, 38, 43, 45, 79b, and 81) were designed for a proper MRD interpretation. Peripheral blood and/or bone marrow specimen were also analyzed and compared with quantitative real-time PCR evaluation of mutation status of IgVH.

In this work, we present results of MRD observation on a pilot group of patients with CLL, who were treated at the Department of Internal Medicine-Hematooncology, University Hospital Brno. The results of treatment responses were correlated with blood count, presence of graft-versus-host disease or influence of immunosupressive therapy. Regular observation of minimal residual disease after the CLL therapy using high-sensitive laboratory methods enables the modification of the therapy before the relapse of the disease and, according to the recent results, it is correlated with the prognosis of the patient.

This work was supported by grant IGA MZ ČR No. NS 9671-4.

Flow cytometeric analysis of T regulatory cells in monoclonal gammopathy patients

Muthu Raja K. R.^1,2, Zahradova L.², Mikulkova Z.^1,4, Kovarova L.², Buresova I.², Hajek R.^2,3, Michalek J.^1,4

¹Dept of Molecular and Cellular Biology, Faculty of Science, Masaryk University, Brno, Czech Republic; e-mail: rajakarthick594@gmail.com

²University Research centre- Czech Myeloma Group, Masaryk University, Brno, Czech Republic

³Internal Hematooncology Department, Faculty Hospital, Brno, Czech Republic

⁴University Cell Immunotherapy Center, Masaryk University, Brno, Czech Republic

Regulatory T cells (T reg) are immunosuppressive T cells which aid in maintenance of self tolerance and comprises about 5–10% among CD4+ T cell population. Monoclonal gammopathy patients were reported to highly associate with immune dysfunctions. So, we were interested to demonstrate the role of T reg cells in monoclonal gammopathy patients.

T reg cells were identified by the surface staining for CD4, CD25, CD127 and followed by intracellular staining for FoxP3. The phenotype of T reg cell population was characterized as CD4+CD25hi+FoxP3+ CD127 low/negative. Analyses of T reg cell level were done in peripheral blood and bone marrow of 68 monoclonal gammopathy patients and peripheral blood of 12 healthy volunteers. The patient group includes 11 MGUS patients, 5 asymptomatic myeloma (AMM) 39 myeloma patients (MM) and 13 relapsed patients. The median age of the patient group was 66.5 years; both male and female patients were in equal proportions in the group. Forty- nine patients had < 10% of bone marrow plasma cells and rest of the patients had > 10% of bone marrow plasma cells. Myeloma patients > 65 years were treated by low-dose regimen (cyclophosphamide 50 mg p.o daily, thalidomide 100 mg daily and dexamethosone 20 mg day 1–4 and 15–18) and < 65 years patients treated by high-dose regimen (cyclophosphamide 500 mg i.v day 1 and 15, thalidomide 200 mg daily, dexamethasone 40 mg day 1–4 and 15–18). Thirty myeloma patients hare been analyzed for T reg cells in peripheral blood after each treatment cycle.

Statistically significant difference was observed in peripheral blood T reg cell level of the respective groups (MM vs relapsed myeloma p = 0.01, relapsed myeloma vs A MM p = 0.01, relapsed myeloma vs MGUS p = 0.01, relapsed myeloma vs healthy volunteers p < 0.01). Bone marrow T reg cell level also showed statistically significant difference between MM and MGUS patients (p = 0.03). No statistically significant difference was noticed in the level of T reg cells between > 10% and < 10% of bone marrow plasma cell infiltration group. Follow-up of myeloma patients during the treatment cycles showed statistically significant data in peripheral blood T reg cell level, between 2nd and 5th cycle (p = 0.04)of low dose group. No significant difference was observed in high dose group.

Our studies showed that there is a trend in increase of T reg cell levels in relapsed myeloma patients than other patient groups and healthy volunteers. There is no significant change in T reg cell levels after treatment, exceptionally in few cases.

This work was supported by MSMT LC06027, MSM 0021622434, MSMT NPVII 2B06058.

Flow cytogenetics accelerates genomics of cereals with complex genomes

Doležel J., Šimková H., Šafář J., Suchánková P., Bartoš J., Kubaláková M., Valárik M., Číhaliková J.

Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, AS CR, v.v.i., Olomouc, Czech Republic; e-mail: dolezel@ueb.cas.cz

Genome analysis and sequencing in barley, rye and wheat is hampered by their large and complex genomes, which consist mainly of repetitive DNA. Polyploidy adds another layer of complexity due to the presence of two and three homoeologous genomes in tetraploid and hexaploid wheat, respectively. While these features make wheat a suitable model to study genome expansion and the role of polyploidy in plant genome evolution, they make genome sequencing and dissection of traits of interest very difficult. With the aim to simplify the analysis of the giant genomes of cereals, we developed strategies to isolate chromosomes and chromosome arms using flow cytometric sorting. This approach makes it possible to target defined parts of the genomes representing only a few percent of nuclear genomes. Particular chromosomes are isolated by sorting based on intrinsic differences in DNA content. However, due to small differences in DNA content, only chromosome 3B can be sorted in wheat. We have demonstrated that this difficulty can be overcome by means of cytogenetic stocks. Thus, in tetraploid wheat, all chromosome arms can be sorted from lines carrying telocentric chromosomes (telosomes). The use of double ditelosomic lines facilitates simultaneous sorting of long and short arms. In hexaploid wheat, all chromosome arms can be sorted from telosomic lines except of long arms of chromosomes 3B and 5B, which can only be sorted as isochromosomes. In barley and rye, only chromosomes 1H and 1R can be sorted from lines with normal karyotypes. However, arms of all remaining chromosomes can be sorted from wheat-barley and wheat-rye chromosome arm addition lines.

The ability to obtain high-quality DNA from particular chromosomes and arms has opened new avenues for genome analysis in these crops. For wheat and rye, chromosome-specific BAC libraries are being created and used to construct BAC contig maps to facilitate genome sequencing and positional gene cloning. The plan is to construct BAC libraries from all 21 chromosomes of hexaploid wheat. Subgenomic BAC libraries and DNA from sorted chromosomes have been used to develop DNA markers to enrich genetic maps at selected genome regions. In applications that require large amounts of DNA but not of high molecular weight, time consuming chromosome sorting can be replaced by representative whole-genome amplification. The amplified DNA can be used for high-throughput allocation of DNA markers to chromosomes and arms using DNA arrays. Shotgun sequencing using the next-generation sequencing technologies is an emerging and powerful application of DNA amplified from sorted chromosomes, as large amounts of sequence data can be obtained from target genome regions in a short time.

This work has been supported by the Czech Science Foundation (grant awards 521/07/1573, 521/08/1629) and the Czech Republic Ministry of Education, Youth and Sports (grant award LC06004).

Structural changes of the chromatin during protoplastization of the plant cells and their regeneration

Ondřej V., Protivánková I., Doležalová I., Lebeda A.

Department of Botany, Faculty of Science, Palacký University in Olomouc, Czech Republic; e-mail: vladan.ondrej@upol.cz

Plant protoplasts (plant cells devoid of a cell wall) represent a very efficient experimental model for physiological, molecular, and developmental studies because of their capability to dedifferentiate, re-enter the cell cycle, and then proliferate or regenerate into the various organs, or else make new plants in the same way as zygotes. They are also valuable tools for biotechnological applications such as somatic hybridization, increasing genetic variability by somaclonal variability, and genetic transformation. It has become a ‘hot topic’ to study protoplast dedifferentiation, not only as a biotechnological application in modern breeding systems, but also in order to find the mechanisms of protoplast development to totipotency and relationships to changes in nuclear organization.

Chromatin is a substrate for several key processes related to the switching on and off of genes within cell differentiation, and development of the organism. The chromatin, in both animal and plant cells, undergoes epigenetic changes, such as in chromatin condensation (Tessadori et al., 2007) and covalent modifications in DNA and in histone tails (Jenuwein and Allis, 2001; Verbsky and Richards, 2001). Protoplast dedifferentiation is characterized by a new balance between the less-dense portion of the genome that is transcribed (euchromatin), and that which is condensed with repressed transcription (heterochromatin). In plants, except those with high genome size (reviewed in van Driel and Fransz, 2004) highly condensed chromatin is easily recognized by light microscopy, after DAPI staining of the interphase nuclei. These blocks of heterochromatin (chromocenters) lose their compaction during protoplast dedifferentiation (Tessadori et al., 2007; Ondrej et al., 2009). Using FISH technique we were able to study rearrangement of the repeated sequences of the cucumber genome that are located in the chromocenters in relation to the protoplasts re-entering to the cell cycle and their regeneration. We found large-scale relaxation of the pericentromeric repeats and subtelomeric satellite DNA type I repeats but just partial relaxation of 5S rDNA repeats. The re-entry to the cell cycle of the protoplasts is demonstrated by the occurrence of partial repeats reassembly and establishing new formed chromocenters 48 hours after protoplasts isolation.

This work was supported by the Ministry of Education of the Czech Republic (MSM 6198959215).

References

1. Jenuwein T, Allis CD. Translating the histone code. Science 2001; 293: 1074–1080.

2. Ondrej V, Kitner M, Dolezalova I, Nadvornik P, Navratilova B, Lebeda A. Chromatin structural rearrangement during dedifferentiation of protoplasts of Cucumis sativus L. Molecules and Cells 2009; 27: 443–447.

3. Tessadori F, Chupeau MC, Chupeau Y, Knip M, Germann S, van Driel R, Fransz P, Gaudin V. Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells. Journal of Cell Science 2007; 120: 1200–1208.

4. van Driel R., Fransz P. Nuclear architecture and genome functioning in plants and animals: what can we learn from both? Experimental Cell Research 2004; 296: 86–90.

5. Verbsky ML, Richards EJ. Chromatin remodeling in plants. Current Opinions in Plant Biology 2001; 4: 494–500.

Glycerol-based preservation technique of plant tissues for flow cytometric analyses

Kubešová M.^1,2, Kolář F.³, Loureiro J., Těšitel J.³, Trávníček P.^1,2, Suda J.^1,2

¹Department of Botany, Faculty of Science, Charles University in Prague, Prague, Czech Republic; e-mail: kubesovamagdalena@seznam.cz

²Institute of Botany, Academy of Sciences of the Czech Republic, Průhonice, Czech Republic

³Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic

Flow cytometry (FCM) has experienced a massive boom in study of plant population biology and biosystematics in last years. The universal use of FCM in this field is, however, strongly hampered by a need of fresh plant tissues for the analyses. Several techniques of storage of plant material for FCM analyses have been proposed, nevertheless, none of them could be accepted as a sufficient universally applicable method. In particular, the poor results of genome size determination procedures (propidium iodide FCM) of long-term stored samples call for further investigations in this field.

We have studied possibilities of isolated plant nuclei storage in 30% glycerol solution in freeze conditions (Hopping 1993). We have focused on the utility of this originally „intra-laboratory“ procedure for a long-term storage of various plant taxa collected in the field. Moreover, performance of the glycerol procedure has been compared to other routinely used plant storage techniques, i.e. silica-gel dried samples and fresh tissue stored in cold. The first dataset based on 34 species from Papua New Guinea tropical forest revealed the wide applicability of the glycerol storage technique across the angiosperms. Moreover, in most cases this procedure gave the most convenient FCM results from all the storage techniques used (the highest proportion of successful analyses and the lowest CVs within the successful analyses). In particular, the applicability of this technique for the genome-size estimation procedure (propidium iodide FCM) should be highlighted. No degradation of the up to 15 days stored frozen samples has been shown as a general feature. Secondly, a laboratory experiment with six taxa (including three routinely used plant standards) has been conducted in order to further evaluate the temporal change of the glycerol-preserved nuclei and to quantify the possible effect of various preserving techniques on fluorescence shifts in FCM analyses.

In conclusions, the glycerol-based preservation technique of plant nuclei seems to be a convenient (yet not universal) alternative for long-term storage of plant material collected for FCM analyses in some flow cytometer remote areas (e.g. the tropics).

References

1. Hopping ME. Preparation and preservation of nuclei from plant-tissues for quantitative DNA analysis by flow cytometry. – New Zealand Journal of Botany 1993; 31: 391–401.

Morphological and ecological differentiation within Glyceria fluitans-group

Chudáčková H.^1,2, Vít P.², Urfus T.^2,3, Zákravský P.³, Hroudová Z.³

¹Dept. of Botany, National Museum, Prague, Czech Republic; e-mail: hana.chudackova@gmail.com

²Dept. of Botany, Charles University, Prague, Czech Republic

³Dept. of Genetic Ecology, Institute of Botany of Academy of Science of the Czech Republic, Průhonice, Czech Republic

Complexes of closely related taxa often show significant differences in ecological demands whereas their morphological differences might be very small. Knowledge of the differentiation between taxa in both ways can reveal important information about microevolutionary processes in the whole group. Wider geographical distribution can be caused by polyploidization but it can also be influenced by higher level of phenotypic plasticity. We have therefore attempted to assess the possible reasons for differentiation within a model group of four closely related taxa – Glyceria fluitans group.

The Glyceria fluitans group is a complex of closely related taxa that differ, besides ploidy level, also in their European area of distribution and frequency of occurrence. Their distribution areas overlap in the Czech Republic. Some differences in reproductive systems in this group are known thanks to work of British botanist Martin Borrill (Borrill 1956, 1958). Frequency of occurrence seems to correlate with ploidy level, tetraploids (G. fluitans and G. notata) being the more frequent while diploids (G. declinata and G. nemoralis) occur rarely. A wide range of methods have been employed in order to reveal which environmental conditions and characteristics of individual species may be connected to their performance and whether their morphological variation correlates with their ecological amplitude.

The species under study showed differences in habitat range (polyploids having more than one ecological optimum), soil reaction (G. fluitans preferring lower pH whereas G. notata occurrs in alkaline, K+ rich soils), soil trophic levels (the occurrence of G. nemoralis seems to correlate closely with the presence of phosphorus in the soil – unlike G. declinata, which prefers soils low in nutrients), and in species composition of plant communities (this reflects the differences in preferred habitats). Among the diploids, G. declinata inhabits secondary habitats, on contrary to G. nemoralis, while tetraploids differ in their preference of either running or stagnant water. Although G. declinata is distributed sparsely in its native area of distribution, it was currently found spreading as an invasive plant in parts of California.

Using a very efficient method of flow cytometry, the nuclear genome size was established for the first time for most members of this group. Differences in nuclear genome size among diploids were found – unlike in tetraploids, which were indistinguishable. The results are consistent with studies showing that species with larger genomes tend to be limited in their distribution and range of ecological environments they can occupy (G. nemoralis, with the highest 1Cx value, is the rarest species in this group). A biosystematic aproach (DAPI and propidium-iodide flow cytometry, chromosome counting using rapid squash techniques, isoenzyme analyses and multivariate morphometrics) was employed together with measurements of environmental factors such as soil chemistry, plant species community etc.

Frequency of occurrence, morphological variation and ties to abiotic environmental factors were compared in order to assess the influence of various environmental factors on the main characteristics of each species. Multivariate statistics shed some light on the main differences between the studied species. Two taxa with previously unknown morphology were found on one location in the vicinity of Znojmo (southern Moravia). As the flow cytometry analyses revealed major differences in these two taxa’s nuclear genome size unlike any interspecies variation found among the rest of the 600 plants, they are thought to be somewhat different from the rest of the Glyceria fluitans group. When morphological differences were found to support the flow cytometry results, a hybrid origin of at least one of these taxa was proposed.

The possibility of allotetraploid origin of Glyceria notata can also be suggested as this species carries intermediate morphological characteristics of the two possible parents – the diploids G. nemoralis and G. declinata. Although the cytometry analyses support this hypothesis we were unable to obtain verification by the means of isoenzyme analyses. Therefore employment of molecular techniques such as AFLP or ITS sequencing is needed in order to solve this matter without any doubts and is expected to be carried out in a near future.

This work was supported by the Grant Agency of the Charles University (project no. 43-257124; 2007-2009).

References

1. Borrill M. A biosystematic study of some Glyceria species in Britain. 1. Taxonomy. Watsonia 1956; 3: 291–298.

2. Borrill M. A biosystematic study of some Glyceria species in Britain. 2. Cytology. Watsonia 1956; 3: 299–306.

3. Borrill M. A biosystematic study of some Glyceria species in Britain. 3. Biometrical studies. Watsonia 1958; 4: 77–88.

4. Borrill M. A biosystematic study of some Glyceria species in Britain. 4. Breeding systems, fertility relationships and general discussion. Watsonia 1958; 4: 89–100.

The Juncus bufonius polyploid complex in central Europe

Rooks F.¹, Jarolímová V.², Drábková L.², Kirschner J.²

¹Department of Botany, Faculty of Science, Charles University in Prague, Czech Republic; e-mail: fredrooks@gmail.com

²Institute of Botany, Academy of Sciences of the Czech Republic, Czech Republic

Cytometric ploidy level screening was done in 120 populations of the cosmopolitan Juncus bufonius group, mainly in central Europe. Two polyploid cytotypes, which are sometimes treated separately as J. minutulus and J. bufonius s. str., were detected and considered to be DNA tetraploids and hexaploids with 2C values of 1.18 ± 2.8% pg 2C DNA and 1.84 ± 1.6% pg 2C DNA, respectively. The correspondence between nuclear DNA content and the number of chromosomes was verified by chromosome counting, establishing that true polyploidy, as opposed to agmatoploidy, is behind the karyological variation. To asses the utility of supposedly diagnostic quantitative morphological characters, measurements of 6 floral and 3 vegetative quantitative characters (no less than 10 measurements per flower, 30 per plant) were obtained for 358 mature plants of known ploidy level from 49 localities. Principal component analysis did not show any separation of the ploidy levels. Canonical discriminant analysis indicated inner tepal length followed by mean capsule width and mean capsule length to be the most useful characters for identifying the ploidy levels; however, the estimated cross-validation error rate of a simple k nearest neighbor classification analysis is 0.45. No novel distinction between the cytotypes was discovered. It is thus concluded that it is impossible to reliably tell apart J. minutulus and J. bufonius s. str. in both mixed or pure populations. Therefore, J. bufonius in Europe is best treated as a single variable species with two cytotypes which are almost inseparable using quantitative morphological traits suggested by extant literature.

Genome size in Hieracium subgenus Hieracium (Asteraceae) is strongly correlated with major phylogenetic groups.

Chrtek Jr. J.^1,2, Zahradnicek J.², Krak K.¹, Fehrer J.¹

¹Institute of Botany, Academy of Sciences of the Czech Republic, Průhonice, Czech Republic; e-mail: chrtek@ibot.cas.cz

²Department of Botany, Faculty of Science, Charles University in Prague, Prague, Czech Republic

Hieracium subgenus Hieracium is one of the taxonomically most intricate groups of vascular plants, due to polyploidy and a diversity of breeding systems including sexuality and apomixis (Gustafsson 1974). The aim of the present study was to analyze nuclear genome size in a phylogenetic framework and to assess relationships between genome size and ploidy, breeding system and selected ecogeographic features.

Holoploid and monoploid genome sizes (C- and Cx-values) of 215 cultivated plants from 89 field populations of 42 so-called ‘basic’ Hieracium species were determined using propidium iodide flow cytometry. Chromosome counts were available for all analyzed plants, and all plants were tested experimentally for their mode of reproduction (sexuality vs. apomixis). For constructing molecular phylogenetic trees, the external transcribed spacer region of nuclear ribosomal DNA was used.

The mean 2C values differed up to 2.37-fold among different species (from 7.03 pg in diploid to 16.67 in tetraploid accessions). The 1Cx values varied 1.22-fold (between 3.51 and 4.34 pg). Variation in 1Cx values between conspecific (species in a broad sense) accessions ranged from 0.24% to 7.2%. Little variation (not exceeding the approximate measurement inaccuracy threshold of 3.5%) was found in 33 species, whereas variation higher than 3.5% was detected in seven species. Most of the latter may have a polytopic origin. Mean 1Cx values of the three cytotypes (2n, 3n and 4n) differed significantly (average of 3.93 pg in diploids, 3.82 pg in triploids and 3.78 pg in tetraploids) indicating downsizing of genomes in polyploids. The pattern of genome size variation correlated well with two major phylogenetic clades which were composed of species with western or eastern European origin. The monoploid genome size in the ‘western’ species was significantly lower than in the ‘eastern’ ones. Correlations of genome size with latitude, altitude and selected ecological characters (light and temperature) were not significant. A longitudinal component was apparent only for the whole data set but was absent within the major lineages.

Phylogeny was the most important factor explaining the pattern of genome size variation in Hieracium sensu stricto, species of western European origin having significantly lower genome size in comparison with those of eastern European origin. Any correlation with ecogeographic variables, including longitude, was outweighed by the divergence of the genus into two major phylogenetic lineages.

References

1. Gustafsson A. Apomixis in higher plants II. The causal aspect of apomixis. Acta Univ Land N. F. Adv 1974; 43: 69–179.

Apoptosis induced by valproic acid in normoxic and hypoxic conditions in neuroblastoma cell lines

Cipro Š.¹, Hřebačková J.¹, Hraběta J.¹, Poljaková J.^1,2, Eckschlager T.¹

¹Dept. of Pediatric Hematology and Oncology, 2^nd Faculty of Medicine, Charles University, Prague, Czech Republic; e-mail: simon.cipro@gmail.com

²Dept. of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic

Valproic acid (VPA) is a widely used histone deacetylase inhibitor. It is well described as a potent inductor of apoptosis in many cancers. Nevertheless, these experiments were performed in normoxic conditions (21% of O₂) only. Commonly, solid tumors contain hypoxic areas that are caused partly by imbalance between angiogenesis and tumor growth and partly by low quality of new vessels. Hypoxia could lead to more aggressive phenotype of tumor cells and contribute to development of chemoresistance. Moreover, hypoxia could up-regulate antiapoptotic proteins such as Bcl-2 or IAP-2 (Dong, 2003) and down-regulate proapoptotic proteins such as Bid and Bax (Erler et al., 2004). Sensitivity of hypoxic cells to apoptosis could be then significantly decreased. Some of these pro/antiapoptotic proteins are under transcriptional control of Hypoxia–Inducible Factor 1 (HIF-1).

HIF-1 is a transcription factor playing pivotal role in adaptation to hypoxia, its protein level is decreased by VPA and other histone deacetylase inhibitors as well (Kim at al., 2007). Thus, we elucidated whether VPA-induced apoptosis is present also in hypoxia and if this type of cell death is processed through the same pathways as in normoxia.

Four neuroblastoma cell lines were used in this study: UKF-NB-3 (N-type), SK-N-AS (S-type) and cell lines derived from these resistant to cisplatin. Apoptosis was measured using two independent methods; sub-G1 peak and annexin V/PI staining (BioVision). Cell cycle analysis was performed with propidium iodide. Cells were evaluated by flow cytometry. For hypoxic conditions, cells were incubated in hypoxic chamber with 1% O₂, 5% CO₂ and 94% N₂. IC₅₀ values of VPA were assessed by MTT assay. Samples were treated with different concentrations of VPA (0.5, 1, 2, 5, 10 mM) for 24 and 48 hours.

We confirmed previous reports that VPA provokes apoptosis in normoxia. Interestingly, we found that VPA-induced apoptosis is much more prominent in hypoxia. These differences between normoxia and hypoxia are evident even after treatment with 1 mM VPA, serum concentration usually found in patients treated with VPA. In UKF-NB-3, relevant amount of apoptotic cells was detected after 24-hour incubation with 2 mM VPA; 3.3 % in normoxia and 10.9 % in hypoxia. Apoptosis in SK-N-AS appeared no earlier than after 48 hours. These findings were consistent with our previous results where in normoxia SK-N-AS cells revealed less susceptibility to VPA (IC₅₀ values was 1.02 mM and 2.06 mM for UKF-NB-3 and SK-N-AS respectively).

We used caspase-8 inhibitor to determine whether apoptosis is initiated by extrinsic apoptotic pathway both in normoxia and hypoxia. 5 mM VPA reduced apoptosis in SK-N-AS cells in normoxia whereas its level remained unchanged in hypoxia. These data suggest that VPA-induced apoptosis is processed through another pathway than in normoxia.

Cell cycle analysis showed that VPA treatment led to G2/M arrest, with significant difference between controls and VPA treated samples at VPA concentrations of 5 and 10 mM. Cell cycle arrest likely contributes to initiation of apoptosis.

To evaluate if the treatment with VPA could prevent chemoresistance in hypoxia, we measured apoptosis induced by 1 mM VPA and 1 μM cisplatin in combination. Rate of apoptosis in UKF-NB-3 treated with cisplatin was 7.5% in normoxia and 4.3% in hypoxia. Treatment of these cells with the combination caused 15.7% apoptosis in normoxia and 18.6% in hypoxia. Our data showed that hypoxia-induced chemoresistance might be prevented by VPA. Moreover, there is synergistic effect between these drugs both under normoxic and hypoxic conditions.

We described that hypoxia does not decrease VPA induced apoptosis which further strengthen the hypothesis that VPA used either in monotherapy or in combination with other anticancer agents could be beneficial in treating neuroblastoma.

This work was supported by grant GAUK 72208/2008.

References

1. Dong Z, Wang JZ, Yu F, Venkatachalam MA. Apoptosis-resistance of hypoxic cells: multiple factors involved and a role for IAP-2. – Am J Pathol 2003; 163: 663–671.

2. Erler JT, Cawthorne CHJ, Williams KJ, Koritzinsky M, Wouters BG, Wilson C, Miller C, Demonacos C, Stratford IJ, Dive C. Hypoxia-Mediated Down-Regulation of Bid and Bax in Tumors Occurs via Hypoxia-Inducible Factor 1-Dependent and Independent Mechanisms and Contributes to Drug Resistance. Mol Cell Biol 2004; 24: 2875–2889.

3. Kim SH, Jeong JW, Park JA, Lee JW, Seo JH, Jung BK, Bae MK, Kim KW. Regulation of the HIF-1 stability by histone deacetylases. Oncol Rep 2007; 18: 647–651.

Reovirus induced cell death in hypoxic and apoptosis resistant medulloblastoma cells

Hraběta J., Figová K., Cipro Š., Eckschlager T.

Department Pediatric Hematology/Oncology, Charles University 2^nd Medical School and University Hospital Motol, Prague, Czech Republic; e-mail: janhrabeta@gmail.com

Despite the advances in clinical oncology that help to decrease patients’ mortality, cancer remains one of the main causes of death in all developed countries. Current efforts to improve cancer therapy are aimed at enhancing drug efficacy while maintaining acceptable degree of toxicity. In order to succeed, innovatory therapeutic modes have been designed. One of these is represented by oncolytic viruses that infect, replicate in, and lyse tumor cells, but do not grow at all, or at a limited extent, in non-tumor cells (Eckschlager and Figová, 2008). Reoviruses (RV) (acronym for Respiratory Enteric Orphan viruses) are viruses that, thanks to their natural properties, selectively replicate in a wide spectrum of tumor cells. Research into the mechanism of reovirus tumor selectivity has revealed that they replicate well in cells with activated Ras signaling pathway, which is an attribute shared by many cancer cells. In vitro studies, animal experiments and, subsequently, clinical studies suggested that reovirus type 3, strain Dearing, may be an efficient and safe anticancer agent (for review see Eckschlager and Figová, 2008).

Tumor hypoxia strongly correlates with advanced disease stage and poor clinical outcome. This is, in part, due to increased genomic instability in hypoxic tumor cells and enhanced resistance of hypoxic tumors to radio- and chemotherapy. While the mechanism of hypoxic cell radioresistance is well characterized, the underlying mechanisms contributing to hypoxia-mediated chemo-resistance are poorly understood. Some studies demonstrated that hypoxia-induced chemoresistance to cisplatin and doxorubicin in vitro is through the HIF pathway and was inhibit by silencing of HIF-1α gene (Brown et al. 2006).

The aim of this study was to compare the effects of RV on medulloblastoma (MBL) derived cell lines in hypoxic and normoxic conditions and to elucidate whether hypoxia (atmosphere with 1% O₂) influence replication of RV and its oncolytic activity in MBL in in vitro conditions.

Used cell lines: medulloblastoma derived lines Daoy and U87. Reovirus type 3, strain Dearing was propagated in Vero cells. Virus stocks were kept frozen at –70 °C. Their titres were determined by the standard plaque assay using agar overlay. For induction of hypoxia conditions in vitro, we cultivated the cells in a closed system (Hypoxic chamber, Billups-Rothenberg, Inc., USA) with defined gas mixture: O₂ (1%), CO₂ (5 %), N2 (94 %). Hypoxic phenotype of tested cells was verified by the expression of HIF protein using Western blot or real time RT PCR (VEGF, GLUT1). We determined RV antigen producing cells by indirect immunofluorescence measured by flow cytometry after permeabilisation of the cells using Fix & Perm kit (An der Grub) according to manufacturer’s protocol. As the primary antibody we used MAB994 monoclonal antibody reactive with RV type 3 σ1 hemaglutinin (Millipore, Billerica, MA) and as the secondary antibody the FITC-Conjugated Goat Anti-mouse Immunoglobulin Polyclonal Antibody (BD). IC₅₀ of cytostatics and RV were assess by MTT test and calculation done from non-linear regression curve. Cell cycle analysis was performed by flow cytometry (FACSCalibur). Apoptosis was determined by flow cytometry as hypodiploid peak and by the examination of modifications of nuclear morphology using fluorescent microscope.

RV is able to replicate in MBL cell line both in normoxic and hypoxic conditions. Percentage of apoptotic cell is higher in normoxia than in hypoxia 24 and 48 hrs after infection however after 72 hrs is higher in hypoxia. We suppose that after 3 days cells are adapted to hypoxia. On the other hand susceptibility of MBL cells to cisplatin is reduced in hypoxia even after 4 days. RV induced arrest of MBL cells in G2/M phase of cell cycle in normoxic conditions and abolished hypoxia induced G0/G1 cell cycle arrest and induced G2/M arrest. This is in accordance with the observations of Tyler and colleagues made in mouse L929 cells and other cell lines susceptible to RV, that the virus induces apoptosis and cell cycle arrest in G2/M phase (Tyler et al, 2001). Pancaspase inhibitor (Z-VAD-FMK) suppressed apoptosis caused by RV in normoxia and hypoxia, but did not significantly increase cell viability.

We conclude that RV induces changes of cell cycle kinetics and death of MBL cells which is caspase independent in normoxic and hypoxic conditions. One might speculate that RV therapy is promising therapeutic tool in MBL which is active also in hypoxic parts of tumor that are less sensitive to radiotherapy and chemotherapy.

Supported by the Ministry of Education, Youth and Sports of The Czech Republic - Grant MSM 0021620813.

References

1. Brown LM, et al. Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor-1. Mol Pharmacol 2006; 69: 411–418.

2. Eckschlager T, Figova K. Reolysin. Drugs future 2008; 33: 489–495.

3. Tyler KL, et al. Reoviruses and the host cell. Trends Microbiol 2001; 9: 560–564.

Expression changes in neuroblastoma cell lines caused by development of drug-resistance

Procházka P.¹, Libra A.², Hraběta J.¹, Poljaková J.¹, Bunček M.², Eckschlager T.¹

¹Department of Pediatric Hematology and Oncology, 2^nd Medical School Charles University and University Hospital Motol, Prague, Czech Republic; e-mail: proch.pavel@gmail.com

²Generi-Biotech, Hradec Králové, Czech Republic

One of the main causes of chemotherapy failure is the rise of resistance. Drug-resistant lines, which can be prepared by exposing parental cells to increasing concentrations of a particular drug, have been shown to be suitable models for the study of changes accompanying resistance and its mechanisms (Kotchetkov et al., 2005). In our work, we concentrated on drug-resistance of high-risk neuroblastoma (HR NBL), which is the most frequent extra-cranial tumor in children and is a major cause of death from neoplasia in infancy.

Drug-resistance in NBL might be caused by multiple factors, including overexpression of the genes for the multidrug-resistance-associated protein (mrp) and MDR1gene-encoded P-170 glycoprotein (Pgp). ATP binding cassette transporters (ABC transporters) could be overexpressed and thus expulse drugs from cells. Drug-resistance could be also caused by changes in apoptotic pathways e.g. mutations of p53 and/or overexpression of Bcl-2 that contribute to neoplastic transformation by blocking apoptosis.

The well known mechanism of drug-resistance is overexpression of Pgp. We used flow cytometry and examined expression of Pgp in cell lines resistant to drugs commonly used in NBL therapy i.e. doxorubicin (DOXO) and vincristine (VCR) and to not common drug in cancer treatment – ellipticine (ELLI). DOXO and VCR-resistant cell lines have high expression of Pgp, which certify that DOXO and VCR are expulsed from cells by Pgp. ELLI-resistant cell line have surprisingly decreased expression of Pgp. In examination of other proteins that could have role in drug-resistance, only mrp2 and CD57 have approximately 1,5× higher expression in comparison to the sensitive cell line.

For better description of possible mechanism of ELLI-resistance we used expression microarray analysis. We registered the downregulation of P-gp expression which was consistent with flow cytometric analysis. The expression of most other ABC transporters was not influenced. In apoptotic pathways, we found deletion of BAX gene investigated by fluorescent in situ hybridization (FISH) and higher expression of Bcl-2 protein. Both have no significant changes on mRNA expression levels. This is not too meaning change and we suppose that modifications in apoptotic pathways might be additional mechanism in ELLI-resistance.

Characteristic change for ELLI-resistance is downregulation of topoisomerase II (TOPII) (Khélifa et al., 1999). TOPII mediate topological changes in DNA bya process in which DNA segments are passing through transientDNA double strand breaks. In microarray and real-time PCR experiment we observed downregulation of both TOP (TOPI and TOPIIα). Downregulation of TOPI and TOPIIα correspond with loss of one gene copy of TOPI and TOPIIα investigated by FISH.

Mechanism of ELLI and DOXO action is similar. Both drugs intercalate DNA and/or induce inhibition of TOPII (Fosse et al., 1992). DOXO-resistant cell line has high Pgp expression which corresponds to upregulation of MDR1. Using expression microarray we found out in DOXO-resistant cell line many expression changes which could fill in next information about DOXO-resistance. DOXO-resistant cells upregulate DNAJC15 which is usually downregulated in drug-resistant cells or his inactivation is connected with development of pediatric brain tumors (Lindsey et al., 2006). Next identify target of drug-resistance is upregulation of MM-TRAG (MGC4175) which is overexpressed especially in DOXO-resistant cells (Duan et al., 2004).

Our results demonstrate that drugs with similar mode of action could have different mechanism of resistance. We confirmed that DOXO- and VCR-resistance is especially caused by overexpression of Pgp in majority of NBL lines. Contrary wise DOXO-resistant MR-32 does not overexpress Pgp. ELLI-resistance is complicated phenomenon which still waiting for future discovery.

This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM0021620813) and Ministry of Health (grant 1A8696-4/2005).

References

1. Duan Z, Brakora KA, Seidel MV. MM-TRAG (MGC4175), a novel intracellular mitochondrial protein, is associated with the taxol – and doxorubicin-resistant phenotype in human cancer cell lines. Gene 2004; 340: 53–59.

2. Fosse P, Rene B, Charra M, Paoletti C, Saucier JM. Stimulation of topoisomerase II-mediated DNA cleavage by ellipticine derivatives: structure-activity relationship. Mol Pharmacol 1992; 42: 590–595.

3. Khélifa T, René B, Le Mée S, Lambert B, Saucier JM, Markovits J, Jacquemin-Sablon H, Jacquemin-Sablon A. Transfection of 9-hydroxyellipticin-resistant Chinese hamster fibroblasts with human topoisomeraseIIalpha cDNA: selective restoration of the sensitivity to DNA religation inhibitors. Cancer Res 1999; 59: 4927–4936.

4. Kotchetkov R, Driever PH, Cinatl J, Michaelis M, Karaskova J, Blaheta R, Squire JA, Von Deimling A, Moog J, Cinatl J Jr. Increased malignant behavior in neuroblastoma cells with acquired multi–drug resistance does not depend on P-gp expression. Int J Oncol 2005; 27: 1029–1037.

5. Lindsey JC, Lusher ME, Strathdee G, Brown R, Gilbertson RJ, Bailey S, Ellison DW, Clifford SC. Epigenetic inactivation of MCJ (DNAJD1) in malignant paediatric brain tumours. Int J Cancer 2006; 118: 346–352.

The minimal residual disease is an independent negative prognostic factor in colorectal cancer patients

Srovnal J.¹, Kesselova M.¹, Vyslouzil K.², Skalicky P.², Straznicka J.³, Sramek V.³, Cwiertka K.³, Ruzkova V.¹, Radova L.¹, Hajduch M.^1,3

¹Laboratory of Experimental Medicine, Department of Pediatrics; e-mail: josef.srovnal@seznam.cz

²1^st Department of Surgery

³Department of Oncology, Faculty of Medicine and Dentistry, Palacký University, and University Hospital in Olomouc, Czech Republic

Minimal residual disease (MRD) in colorectal cancer patients means the presence of isolated tumor cells in patient, who underwent radical resection of primary tumor and has no signs of systemic disease. It is supposed, that these isolated tumor cells can be a precursors of micrometastasis. But prognostic value of MRD is still not clear. In our project we aimed on the detection of MRD in colorectal cancer patients using QRT-PCR method and we correlated our results with clinical-pathological features of the disease.

For the detection of MRD in peripheral blood and bone marrow we used QRT-PCR for a carcinoembryonic antigen (CEA) and cytokeratine 20 (CK20) as a cancer cells markers. This method is very sensitive and can detect 1 cancer cell in nearly 10 millions of non-cancer cells, thus allow us to make more precise staging in colorectal cancer patients. In the years 2004-2006 we examined for MRD more then 200 colorectal cancer patients of stage I-III, who underwent curative surgery at 1^st Department of Surgery of FN and LF Olomouc. The samples of peripheral blood and bone marrow at surgery and one month after surgery were analyzed for the MRD presence in each patient.

We found out, that the bone marrow is more suitable compartment for MRD investigation than peripheral blood. The presence of MRD in bone marrow predicts the systemic relaps. The results of survival analysis show significant better disease-free survival (DFS) in control bone marrow MRD negative patients than MRD positive (p = 0,01). We found, that not only the MRD positivity, but also the increasing of MRD in bone marrow indicates shorter DFS (p = 0.0005).

Conclusion: Using QRT-PCR method we can identify colorectal cancer patients with low clinical stage of disease, but in higher risk of disease recurrence, who could have benefit from adjuvant chemotherapy. In our study we demonstrate, that the minimal residual disease is an independent negative prognostic factor in colorectal cancer patients

This project was supported by grant projects MSM6198959216, IGA MZ CR NR/7804-5 and MPO 1H-PK/45.

Dynamic monitoring of epitelial-mesenchymal transition induced by the transforming growth factor – 1 in prostate epithelial cells

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

¹Department of Cytokinetics; e-mail: ksoucek@ibp.cz 

²Department of Free Radical Pathophysiology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic

Phenomenon of plasticity of differentiated adult cells could have great therapeutic potential, but at the same is characteristic for progression of serious pathological states. Epithelial-mesenchymal transition (EMT) is a crucial process in embryogenesis, but it also occurs during progression of tumors derived from epithelial cells. We report on application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment with the transforming growth factor – 1 in prostate epithelial cells. Changes in the cell index profile were parallel with cytoskeleton rebuilding and induction of epithelial-mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate.

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.

Intracellular lipid droplets in differentiation and/or apoptotic response of colon epithelial cell lines

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

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

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

Lipid droplets (LDs) are found in the cytosol of most eukaryotic cells, where they participate in a variety of cellular functions. They contain a core of triacylglycerols (TAG) and cholesterylesters surrounded by a phospholipid monolayer mainly made up of phosphatidylcholine and specific associated proteins (Olofsson et al., 2008). LDs are reported to be very dynamic organelles, whose appearance changes rapidly upon lipogenesis or lipolysis. LD biogenesis is highly regulated process, that culminate in the compartmentalization of a specific set of proteins and lipids, that place LDs as inducible organelles with role in cell signaling, regulation of lipid metabolism, membrane trafficking and control of the synthesis and secretion of inflammatory mediators (Ducharme et al., 2008). Cells accumulate LDs in response i/ to exogenous lipid availability, present in serum lipoproteins or free fatty acids or ii/ to many kinds of cellular stress, including inflammation, induction of apoptosis by various stimuli or contact inhibition. LD content arising from the medium has a storing purpose for energy generation and membrane building. In the case of stress induction, the metabolic origin of LD-associated neutral lipids and physiological function are not known. LD formation and accumulation is studied mostly in adipocytes and cells of immune system. Less is known about the role of LDs in colon cells and particularly about differences between normal and cancer cells (Accioly et al., 2008).

In our experiments we compared formation of cytoplasmic LDs in human colon epithelial cell lines derived from fetal colon (FHC), adenomas (AA/C1, RG/C2), adenocarcinomas (HT-29, HCT-116), and lymph node metastasis (SW620) to the treatment with short-chain fatty acid - sodium butyrate (NaBt) and ω-3 (DHA, 22:6) or ω-6 (AA, 20:4) polyunsaturated fatty acids (PUFAs) alone or in combination. Accumulation of TAG in LDs was quantitatively measured using flow cytometry (FACSCalibur, BD) and two fluorescent dyes Nile red and BODIPY 493/503. In addition, we searched for the attendance of LD formation with proliferation, differentiation and apoptotic response, which was different in individual cell lines and was accompanied with changes of other parameters such as membrane lipid packing (flow cytometry, merocyanine 540), reactive oxygen species (ROS) production (flow cytometry, DHR-123), number of cells with decreased mitochondrial transmembrane potential (MMP flow cytometry, TMRE), caspase activities (fluorimetry), and expression of regulatory proteins of Bcl-2 family (Western blotting).

NaBt induced G0/G1 or G2/M arrest of the cell cycle, differentiation (FHC, AA/C1, HT-29) and/or apoptosis (RG/C2, HCT-116, SW620) depending on the cell line. Combination of NaBt with AA or especially with DHA suppressed differentiation in FHC, AA/C1 and HT-29 cells and then evoked strong apoptotic response in FHC cells. In these fetal cells, it was associated with more pronounced lipid unpacking, LD accumulation, oxidative response and decrease of MMP than in cancer cells. The role of ROS and caspases was verified by using antioxidant Trolox and pancaspase inhibitor Z-VAD, respectively.

Taken together, our results confirm the interaction of butyrate and PUFAs in colonic cells (Hofmanová et al., 2005) and suggest that changes of lipid accumulation manifested by increased LD formation may be involved in signalling regulating colon epithelial cell kinetic.

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

References

1. Accioly MT, Pacheco P, Maya-Monteiro CM, Carrossini N, et al. Lipid bodies are reservoirs of cyclooxygenase-2 and sites of prostaglandin-E2 synthesis in colon cancer cells. Cancer Res 2008; 68: 1732–1740.

2. Ducharme NA, Bickel E. Lipid droplets in lipogenesis and lipolysis. Encocrinology 2008; 149: 942–949.

3. Hofmanová J, Vaculová A, Lojek A, Kozubík A. Interaction of polyunsaturated fatty acids and sodium butyrate during apoptosis in HT-29 human colon adenocarcinoma cells. Eur J Nutr 2005; 44: 40–51.

4. Olofsson SO, Boström P, Andersson L, Rutberg M, et al. Triglyceride containing lipid droplets and lipid droplet-associated proteins. Curr Opin Lipidol 2008; 19: 441–447.

Dibenzocyclooctadiene lignans restore the cytotoxic action of doxorubicin in multi-drug resistant lung cancer cells COR-L23/R

Slaninová I.¹, Březinová L.², Koubíková L.¹, Hammerová J.¹, Slanina J.²

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

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

Cancer multidrug resistance is one of the major causes of failure of chemotherapy. Over-expression of the ATP binding cassette members, particularly P-glycoprotein (Pgp, ABCB1) and family of multidrug resistance-associated proteins (MRP1 – MRP9) exporting drugs out of the cells, are responsible for most cases of clinical cancer multidrug resistance (Filipits, 2004; Yu et a., 2007). One of the current challenges of anticancer therapy research is uncovering compounds, which can act as MDR modulators and co-administer them with anticancer drugs to make treatment more effective and to minimize drug side-effects. Recently, dibenzocyclooctadiene lignans have been discussed as compounds that have the potential to overcome multidrug resistance (Sun et al., 2007, Pan et al., 2005). These lignans originated from fruit of Schisandra chinensis (Schisandraceae) that is a well-known medicinal plant in traditional Chinese medicine. The fruits and seeds have been used for centuries as a tonic and antitussive. Many studies have indicated that the active ingredients are lignans possessing an unusual structure derived from dibenzo[a,c]cyclooctadiene, which have been shown to possess a broad range of biological effects, including hepatoprotective and antiviral properties. Recently, dibenzocyclooctadiene lignans have been discussed as compounds that are able to overcome multidrug resistance.

In this study, nine dibenzo[a,c]cyclooctadiene lignans, schizandrin, gomisin A, gomisin N, gomisin J, angeloylgomisin H, tigloylgomisin P, deoxyschizandrin, γ schizandrin and wuweizisu C, isolated from seeds of Schisandra chinensis, were examined for their effect on multidrug resistance, as well as their anti-proliferative activities. COR-L23/R, a multidrug-resistant sub-line over-expressing multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) and its parent cell line COR-L23 (human lung cell carcinoma) were used in these experiments. Our observations showed that COR-L23/R was nearly hundred times more resistant to doxorubicin than the parental line COR-L23; although both cell lines were similarly sensitive to lignans treatment, indicating that the lignans are not exported from the resistant cells. We found out that two lignans, R-(+)-deoxyschizandrin and R,S-(±)-γ-schizandrin at relatively non-toxic concentrations restored the cytotoxic action of doxorubicin, a MRP1 substrate, to COR-L23/R cells. Using doxorubicin accumulation assay we demonstrated that R-(+)-deoxyschizandrin and R,S-(±)-γ-schizandrin significantly enhanced the accumulation of doxorubicin in drug resistant cells. Both lignans alone had no effect on the cell cycle; however, when combined with sub-toxic doses of doxorubicin, they induced cell cycle arrest in the G2/M phase, which is typical for toxic doses of doxorubicin.

Our results suggest that R-(+)-deoxyschizandrin and R,S-(±)-γ-schizandrin potentiate the cytotoxic effect of doxorubicin in doxorubicin resistant lung cancer cells COR-L23/R by increasing the accumulation of doxorubicin inside the cells. In the structural point of view, we can conclude that the common structural feature of both active lignans is the R-biaryl configuration and the absence of a hydroxy group at C-8. Unlike the reversal effect, the cytotoxicity of lignans with the R-biaryl configuration was similar to that observed for lignans with the S-biaryl configuration.

This work was supported by the Czech Science Foundation (project No. 522/07/0995) and the Ministry of Education of the Czech Republic (VZ MSM0021622415 and LC06077).

References

1. Filipits M. Mechanisms of cancer: multidrug resistance. Drug Discovery Today: Disease Mechanisms 2004; I: 229–234,

2. Pan QR, Tao W, Lu QH, Hu X. Schisandrin B – A novel inhibitor of P-glycoprotein. Biochemical and Biophysical Research Communications 2005; 335: 406–411.

3. Sun M, Xu XL, Lu QH, Pan QR, Hu X. Schisandrin B: A dual inhibitor of P-glycoprotein and multidrug resistance-associated protein 1. Cancer Letters 2007; 246: 300–307.

4. Yu XQ, Xue CC, Wang G, Zhou SF. Multidrug resistance associated proteins as determining factors of pharmacokinetics and pharmacodynamics of drugs. Current Drug Metabolism 2007; 8: 787–802.

Prediction of localization and interactions of apoptotic proteins

Vařecha M., Ulman V., Matula P., Kozubek M.

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

During apoptosis several mitochondrial proteins are released. Some of them participate in caspase-independent nuclear DNA degradation, especially apoptosis-inducing factor (AIF) and endonuclease G (endoG). Another interesting protein, which was expected to act similarly as AIF due to the high sequence homology with AIF is AIF-homologous mitochondrion-associated inducer of death (AMID). We studied the structure, cellular localization, and interactions of several proteins in silico and also in cells using fluorescent microscopy. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the lipid membranes. Bioinformatic predictions were conducted to analyze the interactions of the studied proteins with each other and with other possible partners. We conducted molecular modeling of proteins with unknown 3D structures. These models were then refined by MolProbity server and employed in molecular docking simulations of interactions. Our results show data acquired using a combination of modern in silico methods and image analysis to understand the localization, interactions and functions of proteins AMID, AIF, endonuclease G, and other apoptosis-related proteins.

This work was supported by The Ministry of Education, Youth and Sports of Czech Republic (project numbers MSM0021622419, 2B06052, and LC535).

Three-color flow cytometric analysis of viability and apoptosis in non-fixed preparations and their morphological aspects

Borský M., Ionescu M., Klabusay M.

Laboratory of Flow Cytometry and Cellular Therapy, University Hospital Brno, Czech Republic, e-mail: mborsky@fnbrno.cz 

Cell viability monitoring is crucial for work and research in the field of tissue culture. For this purpose, easy, fast and reliable assay is required. Flow cytometry can detect apoptosis and necrosis stages by analyzing light-scattering properties of the cells and their surface and intracellular markers. Many assays using different approaches are available. We established innovated three-color flow cytometricprotocols for cells growing in suspension.

Calcein/AnnexinV/PI (CAP) assay assesses intracellular esterase activity of cells by enzymatic conversion of the nonfluorescent cell-permeant calcein AM to intensely fluorescent calcein. Calcein (C) is well retained within a live cell and produces intense uniform green fluorescence (ex/em ~495 nm/~515 nm). Apoptotic cells are distinguished by Ca²⁺ dependent Annexin V - DY647 (ex/em ~633 nm/~672 nm) binding phosphatidylserine (PS) exposed on the outer leaflet of the plasma membrane. Membrane permeability is tested by propidium iodide (PI) (ex/em ~493 nm/~632 nm) that binds stoichiometrically to double-stranded nucleic acid, allowing fluorescence intensity to be used as an indicator of DNA content. Intact cells are PI^- because membrane of living cells excludes cationic dyes, such as PI.

We have used CAP protocol for assessing viability and apoptosis of SU-DHL4 (human B cell lymphoma) and human B-cells by flow cytometry. We observed that this CAP assay describes the apoptosis of cells very precisely. After treatment with an apoptotic activator Campthothecin, six stages of cell fate were distinguished gradually in time. To analyze morphology of these stages we performed cell sorting and staining by Giemsa-Romanowski for light microscopy study.

Intact and viable cells provided C^brightA^-P^- signal and formed compact population on scatter plot. C^brightA^dimP^- cells had higher FSC and morphologically we observed nuclear shrinkage with (or without) small nuclear fragments in basophilic cytoplasm. Cells with lower FSC parameter and two or more nuclear fragments in acidophilic cytoplasm were situated in to the gate C^brightA⁺P^- while gates C⁺A⁺P^- and C^dimA⁺P^- included cells with blebs, progressed karyorhexis and strong acidophilic cytoplasm. Finally, C^-A⁺P⁺ cells were found in gate FSC^dimSSC^bright and their edges were rimless with different numbers of nuclear fragments in strong acidophilic cytoplasm. The last stage of cell’s fate appears after disintegration of cells onto apoptotic bodies in gate FSC^-SSC^-. Calcein positive (A^-P^-) cytoplasm fragments of SU-DHL4 cells were noticed in the same gate as well. Theoretically we can suggest others categories like C^bright/+A^dim/+P⁺ (injured cells) and C^-A^-P⁺ (free nuclei).

Compared to other methods, CAP assay uses positive markers for three stages of the cell fate and provides more information. However, it cannot detect very early apoptosis. Although we described in detail viable and apoptotic stages of SU-DHL4 and B-lymphocytes by CAP assay and morphological analysis, it is necessary to optimize this protocol for other kinds of cells and cytometers.

This work was supported by grant IGA NR9670-4.

References

1. Krysko DV, Berghe TV. Apoptosis and necrosis: Detection, discrimination and phagocytosis. Methods 2008; 44: 205–221.

2. Huerta SMD, Goulet EJ. Screening and Detection of Apoptosis. Journal of Surgical Research 2007; 139: 143–156.

Combined chemo-immunotherapy for the treatment of hormone-refractory metastatic prostate cancer

Rožkov, D.¹, Tišerová H.¹, Fučíková J.¹, Lašt’ovička J.¹, Podrazil M.¹, Ulčová H.¹, Budinský V.¹, Prausová J.², Linke Z.², Minárik I.^1,3, Šedivá A.¹, Špíšek R.¹, Bartůňková J.¹

¹Institute of Immunology, Charles University, 2^nd Faculty of Medicine, University Hospital Motol, Prague, Czech Republic; e-mail: daniela.rozkova@lfmotol.cuni.cz

²Department of Oncology and Radiotherapy, Charles University, 2^nd Faculty of Medicine, University Hospital Motol, Prague, Czech Republic

³Department of Urology, Charles University, 2^nd Faculty of Medicine, University Hospital Motol, Prague, Czech Republic

Immunotherapy has emerged as another treatment modality in cancer. The goal of immunotherapy in advanced cancer patients does not have to be the complete eradication of tumor cells but rather the restoration of a dynamic balance between tumor cells and the immune response. Appropriate combination of tumor mass reduction (by surgery and/or chemotherapy) and neutralization of tumor-induced immunosuppression might set the right conditions for the induction of anti-tumor immune response by active immunotherapy.

More than four hundred prostate cancer patients have been treated with DC-based immunotherapy and tumor-specific immune responses have been reported in two-thirds of them. In half of these patients, DC immunotherapy resulted in transient clinical responses.

We review experimental basis and key concepts of combined chemo-immunotherapy and document its principles in the case report of patient with hormone refractory metastatic prostate cancer with sinister prognosis.

Tregs, among other factors, potently inhibit tumor-specific T cells. Prostate cancer patients have elevated numbers of circulating and tumor infiltrating Tregs and there is evidence that Tregs increase tumor growth in vivo. We measured the Tregs levels and because of the high frequency of circulating Tregs in our patients, we first administered metronomic cyclophosphamide. After obtaining IRB approval, we started regular vaccinations with antigen pulsed autologous dendritic cells (DCs). DCs were generated from patient monocytes cultivated in the presence of cytokines. Immature DCs were then cocultivated with apoptotic prostate cancer cells (UV-treated allogennic cancer cell line LNCap) and their phenotype as well as the pulsation efficiency was analyzed by multicolor flow cytometry.

Overall six doses of dendritic cells-based vaccine have been administered every 3–4 weeks, subsequently every 6 weeks. In accordance with the principles of combined immunotherapy, we continued palliative chemotherapy with docetaxel to reduce the tumor cell burden. DC-based vaccination induced prostate cancer cell-specific immune response. Combined chemo-immunotherapy consisting of alternate courses of chemotherapy and vaccination with mature DCs pulsed with LNCap prostate cancer cell line led to the marked improvement in the clinical and laboratory presentation and to the decrease of PSA levels by more than 90%.

Polyfunctional CMV-specific T-cell response but not IFN-γ+ alone is a hallmark of protection from CMV reactivation in children post-transplant

Krol L.^1,2, Kotus D.², Hubacek P.¹, Sedlacek P.¹, Kalina T.1,2

¹Department of Pediatric Hematology and Oncology, Charles University 2^nd Medical School and University Hospital Motol, Prague, Czech Republic; e-mail: ladislav.krol@fnmotol.cz

²CLIP (Childhood Leukaemia Investigation Prague) – Cytometry, Prague, Czech Republic

Cytomegalovirus (CMV) infection causes significant morbidity and mortality in children after hematopoietic stem cell transplantation (HSCT). In order to find hallmark of protection we studied the functional composition and magnitude of CMV-specific T cell response in CMV-seropositive (CMVpos) and CMV-seronegative (CMVneg) children and compared it to healthy adults and fully reconstituted patients post-HSCT. The response profile was compared to patients with CMV reactivation.

Polychromatic flow cytometry was used for detection of CMV-specific immune. The ability of CD4⁺ and CD8⁺ T-cells to produce cytokines: interferon-γ (IFN-γ) and interleukin-2 (IL-2) and to express activation marker CD40L and/or to mobilize the degranulation marker CD107a in response to CMV antigens was evaluated by intracellular cytokine staining method after in vitro stimulation. Peripheral blood samples were collected from 10 CMVpos children, 10 CMVneg children, 10 CMVpos adults and 10 pediatric patients who underwent HSCT 2 or more years before. We also evaluated 14 samples from 3 patients with detectable CMV viremia who were treated for CMV reactivation by ganciclovir.

The type of polyfunctional CMV-specific response was evaluated in healthy CMVpos and CMVneg children. The comparison showed significant (p < 0.01) CMV-specific response was marked by CD4⁺ production of IFN-γ+IL-2+154+ (mean 0.077%) and IFN-γ+154+ (mean 0.09%) and by CD8⁺ production of IFN-γ+IL-2+ (mean 0.028%), IFN-γ+107a+ (mean 0.073%) and IFN-γ+ (mean 0.45%). CMV-specific responses of adults were of the same type, but the magnitude of the response tended to be higher. Finally, we compared CMV-specific responses in CMVpos children and pediatric patients post-HSCT. In HSCT patients all CMV responses were several times elevated with maximal 13-fold elevation in CD8+IFN-γ+ subset. However, the monofunctional CD8+IFN-γ+ subset is not a specific hallmark of CMV-protection, since all 3 patients post-HSCT with ongoing CMV reactivation had increased numbers of these cells over healthy children (mean 0.98%; SD ± 1.801 versus 0.45%; SD ± 0.378).

CMV-specific immune response is determined by a concerted action of polyfunctional CD4⁺ and CD8⁺ T-cells that has similar composition in children and adults, but is markedly increased in children post-HSCT. Monofunctional CD8+IFN-γ+ response doesn’t correlate with protection of patient from CMV reactivations.

Supported by grant GAUK-47807/2007, MZO000064203 and IGA-NS/9996-4.

Human and pig peripheral blood lymphocytes as biodosimetric indicators: a flow cytometric study

Sinkorova Z.¹, Sinkora J.², Vokurkova D.³, Zarybnicka L.⁴

¹Department of Radiobiology, University of Defence, Hradec Králové, Czech Republic; e-mail: sinkorova.zuzana@seznam.cz

²BD Czech Republic, s.r.o, Prague, Czech Republic

³Institute of Clinical Immunology and Allergology, Charles University in Prague

⁴Faculty of Medicine in Hradec Králové, Czech Republic

The aim of this study was to identify peripheral blood lymphocyte subpopulations and subsets with unique radioresistance or radiosensitivity and to study their potential as biodosimetric indicators that would allow for retrospective determination of the received dose of ionizing radiation. To introduce swine as a large animal model in radiobiology for comparison of in vitro data with the situation in vivo.

Heparin-treated porcine or human peripheral blood was irradiated with selected doses of Co⁶⁰γ rays and incubated for predefined time interval in a humidified atmosphere at 37°C. After that multicolor surface immunophenotyping and flow cytometry were used for the determination of lymphocyte subset proportions. Briefly, blood aliquots were stained with cocktails of fluorochrome-conjugated monoclonal antibodies directed against lymphocyte markers, red blood cells were removed by ammonium-chloride based lyzing solution, leukocytes were washed and data were acquired and analyzed on a CyAn (Beckman Coulter) or FACSCalibur (Becton Dickinson) flow cytometer. In some cases, Ficoll Histopaque isolated peripheral blood mononuclear cells were used. Progression of radiation induced apoptosis was also investigated by fluorinated Annexin V binding to superficially exposed phosphatidyl serine and propidium iodide exclusion assay. For in vivo or ex vivo studies, 4 week-old piglets were irradiated by a selected dose (0–10 Gy) and peripheral blood lymphocyte compartment composition was studies either directly after blood collection (in vivo approach) or after incubation of the collected blood ex vivo. Results: We have demonstrated that lymphocyte shrinking accompanied by a forward scatter parameter value decrease occurs at early stages of apoptosis. A homogenous population of lymphocytes with unaffected scatter characteristics thus consists of a great majority of “intact” cells (lymphoid cells that do not bind Annexin V) and a small fraction of Annexin V-low lymphocytes in the very first stage of apoptosis in terms of phospatidyl serine translocation. Careful gating in the scattergram thus allows for studying cells surviving irradiation in vivo or subsequent cultivation in vitro and no other apoptosis detection is required in large scale experiments. Based on phosphatidyl serine and propidium iodide binding we have designed four stages of apoptosis progression: early, intermediate, late and very late represented by Annexin V^lo PI^-, Annexin V^hi PI^-, Annexin V^hi PI^lo and Annexin V^hi PI^-hi cells, respectively. Relative radiosenstivity of individual lymphocyte subsets was then determined by comparing their proportions among the intact population in irradiated and control, sham-treated samples using the IVNIR (irradiated versus non-irradiated ratio) parameter, which has been calculated as the percentage of the selected subset in the irradiated sample divided by its respective value in the non-irradiated control.

We have proven that markers commonly used in biodosimetric research like CD4 and CD8 are not suitable as biodosimetric indicators due probably to the heterogeneity of the CD4⁺ and CD8⁺ lymphocyte population: each of them consists of cells at different stages of differentiation that possess unequal radiosensitivity. In our hands, well defined, therefore much more homogeneous lymphocyte subsets have proven much more suitable for radiobiological purposes. We have identified human CD8⁺ NK cells, CD21^- B-cells and CD27⁺ B-cells as useful bioindicators of the received dose in vitro. In contrast, essentially all NK cells in pigs have the CD8⁺ phenotype and this marker cannot be used for identification of an unusually radiosensitivite population like in humans. Quite the opposite, CD8⁺ NK cells represent a predominating lymphoid population in sublethally irradiated pigs which can at least partially be explained by mobilization on NK cells upon irradiation and/or maturation of radioresistant NK precursors. In this respect, CD8⁺ NK cells in pig peripheral blood also possess biodosimetric capacity but their behavior has an opposite trend that in human blood in vitro.

Three lymhocyte subsets with biodosimetric potential have been identified in humans; two of them being B-cells and the third one are NK cells bearing CD8 on the surface. Significant interspecies differences have been found between humans and pigs in terms of lymhocyte subset radiosensitivity. On the other hand, swine as a large animal model is very useful for comparing radiobiological experiments in vitro and in vivo and provides a powerful tool for ex vivo (incubation of blood collected from formerly irradiated individuals) data interpretation.

This work was supported by the Ministry of Defense of the Czech Republic (Grant No. OPUOFVZ 200806) and the Ministry of Education, Youth and Sports of the Czech Republic (Grant No. 2B08028).

References

1. Vokurková D, Sinkora J, Vávrová J, Rezácová M, Knízek J, Ostereicher J. CD8+ natural killer cells have a potential of a sensitive and reliable biodosimetric marker in vitro. Physiol Res 2008; 55: 689–698.

2. Rehakova Z, Sinkora J, Vlkova M, Vokurkova D, Osterreicher J, Vavrova J, Driak D. CD27(+) peripheral blood B-cells are a useful biodosimetric marker in vitro. Physiol Res 2008; 57: 589–600.

Profiling of polychromatic flow cytometry data on B-cells reveals patients’ clusters in CVID

Kalina T.^1,2, Stuchlý J.^1,2, Janda A.^1,2, Růžičková Š.³, Litzman J.⁴, Šedivá A.⁵, Vlková M.⁵

¹Department of Pediatric Hematology and Oncology, 2^nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic; e-mail: tomas.kalina@lfmotol.cuni.cz

²CLIP - Childhood Leukemia Investigation Prague

³Laboratory of Diagnostics of Autoimmune Diseases, Institute of Biotechnology of Academy of Sciences of the Czech Republic, p.r.i., Prague, Czech Republic

⁴Department of Clinical Immunology and Allergology, St Anne’s Faculty Hospital and Medical Faculty, Masaryk University, Brno, Czech Republic

⁵Department of Immunology, 2^nd Faculty of Medicine, Charles University Prague and University Hospital Motol,Prague, Czech Republic

The aim of this study was to find an expert-free approach for phenotype analysis of Common Variable Immunodeficiency (CVID) patients that describes all differences in the 6-color space and to form groups of patients with similar phenotypes using computational methods.

CVID is a heterogeneous primary immunodeficiency disorder. The defined molecular defect is recognized in less than 10% of cases and is unknown in the majority of patients. The current CVID classification, EUROClass (1), is based on quantification of selected B-cell subsets.

Using 6-color polychromatic flow cytometry, we analyzed B-cell phenotypes in a cohort of 48 CVID patients and 49 healthy donors. We used a “probability binning” algorithm (2) to create 1024 bins (i.e., six-color gates) that optimally covered the cells’ distribution within the entire B-cell compartment. A matrix file recording cellular content in all the bins was made. The hierarchical clustering of the individual samples was analyzed using a Pearson correlation of the bins’ values.

The Cut Tree algorithm found 12 clusters. In 6 clusters, healthy individuals predominated; in one cluster, smB+CD21low (CVID patients by EUROClass) cells prevailed; in one cluster, smB-CD21norm cells prevailed; in one cluster, smB+CD21low cells prevailed; the remaining cluster was mixed.

The overall reproducibility of this approach was evaluated by testing replicates of two samples from the same donor 98 and 336 days apart. When the B-cell profile of replicates was used to match the original cohort using the similarity matrix of the Pearson correlation, 15 replicates matched the same individual, 3 replicates matched a different individual within the same cluster and 3 replicates matched to a different cluster. We were able to define B-cell subsets over- or under-represented in a particular cluster and display them back in the flow-cytometry software, thus resembling manually created gating.

To conclude, we describe a new analytical approach that enables a search in an expert-free environment for patient cohorts that are defined by similar B-cell profiles and thus contribute to the description of differences between CVID patient groups.

This work was supported by grants IGA NR/9198-3 and MZ CR 000064203.

References

1. Roederer M, Moore W, Treister A, Hardy RR, Herzenberg LA. Probability binning comparison: A metric for quantitating multivariate distribution differences. Cytometry2001; 45: 47–55.

2. Wehr C, Kivioja T, Schmitt C, et al. The EUROclass trial: defining subgroups in common variable immunodeficiency. Blood 2008; 111: 77–85.