ABSTRACTS: Friday 27 March 2015 1. Katerina Jedlickova Empirical antibiotic (ATB) therapy is a standard treatment used in intensive care units[SB1] . Rational antibiotic administration may include two strategies:[SB2] either antibiotic de-escalation (e.g. [SB3] initial empiric [SB4] treatment of antibiotics with a very broad spectrum and subsequent targeted antibiotic treatment with narrower spectrum)[INS: , :INS] or antibiotic escalation ([SB5] an initial choice of fundamental broad-spectrum antibiotics, typically aminopenicillins with beta-lactamase inhibitors, which are changed to antibiotic with a broad spectrum of activity, when found[INS: to be :INS] ineffective). In this retrospective study[INS: , :INS] we analyzed all patients hospitalized in the Dept. [SB6] of Anesthesia and Intensíve Care of St. Anna University Hospital Brno (AIC) in the first quarter of 2013. Patients who had an antibiotic treatment[INS: which lasted :INS] longer than 24 hours and who were hospitalized for more than 48 hours were included in the study. These patients were divided into two groups: the first group was initially treated with aminopenicillins with beta-lactamase inhibitors alone or in [DEL: a :DEL] combination with another antibiotic ("Amino" group). The second group was treated with any other antibiotic as the first choice ("Other ATB" group)[INS: . The :INS] [DEL: F :DEL] [INS: f :INS] ollowing variables were compared: age, Acute Physiology and Chronic Health Evaluation score II (APACHE II), C-reactive protein (CRP) on admission to ICU, CRP at discharge, length of stay and survival. [DEL: :DEL] 2. Vaclav Seda ZAP-70 directly affects BCR signalling and migration of malignant B cells via GAB-1 [INS: A :INS] [DEL: H :DEL] [INS: h :INS] igh expression of ZAP-70 (70-kDa zeta-associated protein) in chronic lymphocytic leukemia (CLL) is associated with enhanced B-cell receptor signalling, migration of CLL cells and worse [SB7] survival of patients. However, the mechanism underlying its function in CLL has not yet been [SB8] well established. For this purpose[SB9] , we investigated whether ZAP-70 can directly affect any proteins involved in BCR activation and AKT phosphorylation and if any of these proteins can alter [INS: the :INS] migration of B cells.[INS: :INS] A total of 37 CLL samples were analysed by Western blot for expression of ZAP-70 and other proteins involved in B cell receptor signalling pathway. Two samples with ZAP-70 higher expression were stimulated with anti-IgM antibody and co-immunoprecipitated. We established that ZAP-70 directly affects [INS: the :INS] expression and function of GAB-1, which is the docking protein involved in B cell receptor signalling. We have observed [SB10] that ZAP-70 high expression in CLL cells positively correlates with the amount of GAB-1 protein (P=0,0003). This aberrant expression of GAB-1 may be caused by protection provided by ZAP-70, which binds [INS: the :INS] GAB-1 protein thus preventing its down-regulation. Furthermore, the dependence of AKT activation on the amount of GAB-1 protein is significant (P=0,0204). In addition, our results indicate that migration potential of MEC-1 cells is significantly reduced after GAB-1 silencing (P=0,0168). [DEL: Taken together :DEL] [INS: When consolidated :INS] , our data indicate that enhanced B cell receptor signalling and AKT activation is caused by aberrant expression of [INS: the :INS] GAB-1 protein in the case of ZAP-70 positive CLL cells. We conclude that the migration of malignant B cells into the supportive microenvironment of stroma cells is affected by changes in [INS: the :INS] expression of GAB-1. [INS: :INS] [DEL: :DEL] 3. Katerina Cerna The biology of B cell Non-Hodgkin lymphomas (NHLs) is largely influenced by [INS: the :INS] (de)regulation of B cell receptor signaling and DNA damage response pathway (DDR). We and other[INS: researcher :INS] s have previously shown that miRNAs are crucial for [INS: the :INS] regulation of these pathways. To investigate the DDR-related miRNAs, primary chronic lymphocytic leukemia (CLL) cells were treated with fludarabine in vitro and paired samples (before/after treatment, n=20) were analyzed for miRNAs’ profile by NGS technology (HiSeq). This identified 6 [SB11] differentially expressed miRNAs (FDR<0.1). To investigate these miRNAs in vivo we profiled the expression of 3[SB12] up-regulated miRNAs (miR-34a, miR-1246, miR-1248) in samples obtained from 50 CLL patients before and during the therapy containing fludarabine (FCR regiment). This confirmed [INS: the :INS] up-regulation of all identified miRNAs (fold change>1.5, P<0.05). Importantly, the miR-34a level was a significant predictor (P<0.05) of patients’ [SB13] response to the FCR therapy and the progression free survival (19.9 vs. 26.4 months; HR: 2.29). A similar trend was observed for miR-1246, however, this was not statistically significant (P=0.11). Additionally, low miR-34a was an independent predictor of a shorter overall survival in a multivariate analysis (16.7 months vs. not reached; P=0.0002; HR: 3.30). This suggests that CLL cells with low levels of miR-34a fail to down-modulate genes that are crucial for DDR. Several pro-survival genes targeted by miR-34a (BIRC3, BCL2, FOXP1) were recently identified in various cell types. These proteins were down-modulated in CLL cells that up-regulate miR-34a during DDR, but not in cells that failed to induce it. Surprisingly, miR-34a, miR-1246 and miR-1248 share numerous validated targets with miR-150, a known negative regulator of BCR signaling (Mraz et al., 2014). This suggests possible convergence in the mechanism of action of DNA damaging drugs and BCR inhibitors recently approved for treatment of NHLs (e.g. ibrutinib). To compare the effects of the FCR with the ibrutinib, we analyzed [INS: the :INS] miRNA/gene expression in samples from ibrutinib treated CLL patients (n=9) before and during the therapy. The convergence of pathways targeted by DDR and BCR inhibition through changes in miRNAs' expression are currently being [DEL: interrogated :DEL] [SB14] [INS: investigated :INS] . [DEL: :DEL] 4. Zuzana Jašková Mutated p53 Protein Elimination and Induction of p53 Downstream Genes in Chronic Lymphocytic Leukemia (CLL) Cells Using PRIMA-1MET In CLL, poor prognosis is associated with missense mutations located in p53 DNA-binding motifs leading to accumulation of [INS: the :INS] mutated p53 protein with oncogenic properties. Thus, the affected patients could benefit from therapy target[INS: ing :INS] [DEL: ed to :DEL] [INS: the :INS] p53-mut protein. We tested a possibility of subtle molecular manipulation with accumulated mutated p53 in CLL cells using PRIMA-1MET declared to change p53-mut to p53-wt conformation with rescue of its activity. CLL patients’ B-cells were chosen from the cohort characterized for TP53 status. Viability was determined by WST-1 assay.[INS: The :INS] [DEL: I :DEL] [INS: i :INS] mpact of PRIMA-1MET on p53 protein level[INS: s :INS] was analyzed by WB. The p53 downstream genes’[SB15] induction was assessed by qRT-PCR. The impact of PRIMA-1MET on CLL cells’ viability was assessed to determine[INS: the :INS] concentration range for reactivation. We observed concentration-dependent viability curves in all samples (n=5) between 0.5-4µM. Therefore, 2µM and 4µM were applied for reactivation testing at molecular level. Five out of eleven mutated samples showed diminished or absent p53 protein after 4µM treatment[INS: , :INS] and four samples manifested partially reduced level[INS: s :INS] . The complete p53 protein elimination at 2µM was observed for mutations p.Y205H, p.R248W, and p.C275R. The p53 downstream genes’ [SB16] induction was pronounced for p21 and GADD45, which were induced 118 and 45-fold, respectively, compared to control. Concerning the specificity of observed effects, we noted the following: (i) in line with other studies, the impact of PRIMA-1MET on p53-wt and p53-mut samples’ viability was similar, and (ii) p53 target genes’ induction was also evident in p53-wt samples. Additionally, we analyzed 10 TP53-mut samples without p53 protein stabilization for downstream genes’ induction. Again, the effects of PRIMA-1MET were apparent, especially for p21 and GADD45.We demonstrated that PRIMA-1MET can effectively manipulate p53 protein accumulated due to the high-risk TP53 mutations. It remains to elucidate, what is the specificity of the p53 pathway activation with respect to TP53 status.[INS: :INS] 5. Veronika Olejnickova Voltage sensitive dye (VSD) represent[INS: s an :INS] important tool for[INS: the :INS] study of cardiac electrophysiology. This touchless method allow[INS: s :INS] simultaneous monitoring action potential of the cardiac tissue with high space and temporal resolution. The VSD mapping is based on excitation of the dye molecules bound to the cell membrane under fluorescence light. Consequently, the excitation of the molecules [SB17] results in [INS: the :INS] emission of [DEL: the :DEL] light and shifting of the emitted light spectrum. The [DEL: spectrum :DEL] [INS: latter :INS] is proportional to the transmembrane potential of the cells. Nevertheless, there is a concern about interference with measured parameters due [INS: to :INS] bounding [SB18] of the dye to the cell membranes. The slowing of cardiac impulse propagation in the presence of the VSD was shown[SB19] . There is no evidence about[INS: the :INS] impact of VDS [DEL: to :DEL] [INS: on :INS] the conduction system of the heart, however [INS: a :INS] slowing of the impulse propagation may subsequently [INS: have :INS] resulted [DEL: to :DEL] [INS: in :INS] disturbances in generation of heart rhythm[INS: s :INS] or to its block, which, in turn may trigger arrhythmias. The aim of this study was therefore [INS: to :INS] evaluate[DEL: d :DEL] [INS: the :INS] influence of the most employed VSD di-4-ANNEPS [DEL: to :DEL] [INS: on :INS] the cardiac conduction system in isolated rabbit heart[INS: s :INS] . The study was perform[INS: ed :INS] in Langendorff isolated rabbit hearts. The electrograms (EGs) [DEL: was :DEL] [INS: were :INS] recorded [DEL: by :DEL] [INS: using :INS] three orthogonal touch-less system[INS: s :INS] . The effect [DEL: to :DEL] [INS: of :INS] the cardiac conduction system was asses[INS: ed :INS] by change[INS: s :INS] in the heart rate (HR), EG morphology and arrhythmogenicity scoring. Perfusion of the di-4-ANNEPS (1µM) leads to the slowing of the HR to 70%. QT and QTc interval, EG morphology was not changed. [DEL: It was observed m :DEL] [INS: M :INS] oderate arrhythmias, mainly AV blocks and single ventricular complexes[INS: , were observed :INS] . [INS: The :INS] [DEL: S :DEL] [INS: s :INS] core of arrhythmogenicity according Lambeth was 0,4. We can conclude that 1µM di-4-ANNEPS slow[INS: s the :INS] heart rate and leads to the disturbance of heart rhythm[INS: s :INS] in isolated rabbit heart. The findings strongly suggest direct impact of the di-4-ANNEPS [DEL: t :DEL] o[INS: n :INS] the conduction system of the heart. [DEL: :DEL] 6. Gabriela Pavlasova Bone marrow stromal cells protect CLL cells from rituximab effects, but do not [SB20] induce down-modulation of CD20 expression. Rituximab, a monoclonal antibody against CD20, is widely used in[INS: the :INS] treatment of B-cell non-Hodgkin lymphomas and chronic lymphocytic leukaemia (CLL). However, it has been shown that microenvironmental interactions protect mature B cell malignancies from rituximab induced apoptosis and complement-dependent cytotoxicity. Therefore, we tested whether bone marrow (BM) stromal cells (HS-5) cause down-modulation of CD20 on CLL surface[INS: s :INS] . We isolated CLL cells from peripheral blood (PB) and cultured them with HS-5 or on plastic alone as a control for 24, 48, 72 hrs [SB21] and 1 week. Flowcytometric analysis of CD20 expression was performed only on Annexin-V neg/CD105 neg cells. We found out [SB22] that CLL cells spontaneously down-modulate CD20 expression when cultured on plastic in vitro, and the co-culture with HS-5 up-regulates CD20 (P=0.03). Additionally, isolated CXCR4dim/CD5bright cells, representing CLL cells that had been recently released from the lymph node (LN) microenvironment, expressed higher surface levels of CD20 when compared to CXCR4bright/CD5dim from the same patient. Th[INS: e :INS] [DEL: i :DEL] s[INS: e :INS] finding[INS: s :INS] suggests that [DEL: also :DEL] the in vivo contact with immune niches [INS: also :INS] up-regulates CD20. Finally, to further investigate the effect of microenvironment on CLL cells in vivo, gene expression profiling of CLL cells purified from PB, BM and LN was performed. [INS: A :INS] [DEL: S :DEL] [INS: s :INS] tatistically significant change in CD20 expression was observed [INS: in :INS] only [DEL: in :DEL] 1 of 4 [SB23] [INS: cases :INS] analyse[DEL: s :DEL] [INS: d :INS] ([SB24] using different probes), [DEL: when :DEL] compar[INS: ed :INS] [DEL: ing :DEL] [INS: with :INS] PB and BM or LN. In this case, the expression of CD20 was slightly higher in BM/LN than PB (P=0.0482; P=0.0495). To sum up, these data indicate that down-modulation of CD20 by bone marrow stromal cells is [INS: un :INS] likely[INS: to be :INS] [DEL: not :DEL] the major mechanism of stroma mediated rituximab resistance of CLL cells. [DEL: :DEL] 7. Katerina Musilova Low-level expression of miR-150 in follicular lymphoma is related to higher B cell proliferation rate and unfavourable disease prognosis We and others have shown [SB25] that deregulation of microRNAs (miRNAs) is associated with the pathogenesis and the progression of B-cell malignancies. Moreover, miRNAs can be used as strong predictors of prognosis in chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). However, their role as prognostic markers in follicular lymphoma (FL) has not [INS: yet :INS] [INS: [SB26] :INS] been investigated[DEL: yet :DEL] . To address this issue[INS: , :INS] we selected three miRNAs (miR-155, miR-181b, miR-150) which play critical roles in B-cell development[INS: : :INS] [DEL: and :DEL] [SB27] their aberrant expression was shown to affect the prognosis of CLL, DLBCL, and MCL patients. We analysed the expression of these miRNAs in 89 FL samples (fresh frozen and formalin-fixed paraffin-embedded [FFPE] tissues). miR-150 expression levels were significantly lower in [DEL: the :DEL] patients with high Ki67 positivity (>20%; P=0.003) and high FLIPI score[INS: s :INS] (3-5; P=0.03). We also observed significantly reduced miR-150 levels in FLs that transformed to DLBCL compared to the samples before transformation (P=0.01), and miR-150 was significantly less expressed in DLBCL than FL (4-fold change, P<0.001). Furthermore, FL patients with low miR-150 levels (