Boris Tichý

miR-34a, miR-29c and miR-17-5p are downregulated in CLL patients with TP53 abnormalities.

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The planar cell polarity pathway drives pathogenesis of chronic lymphocytic leukemia by the regulation of B-lymphocyte migration.

The planar cell polarity (PCP) pathway is a conserved pathway that regulates cell migration and polarity in various contexts. Here we show that key PCP pathway components such as Vangl2, Celsr1, Prickle1, FZD3, FZD7, Dvl2, Dvl3, and casein kinase 1 (CK1)-ε are upregulated in B lymphocytes of patients with chronic lymphocytic leukemia (CLL). Elevated levels of PCP proteins accumulate in advanced stages of the disease. Here, we show that PCP pathway is required for the migration and transendothelial invasion of CLL cells and that patients with high expression of PCP genes, FZD3, FZD7, and PRICKLE1, have a less favorable clinical prognosis. Our findings establish that the PCP pathway acts as an important regulator of CLL cell migration and invasion. PCP proteins represent an important class of molecules regulating pathogenic interaction of CLL cells with their microenvironment.

Intracoronary Delivery of Bone Marrow Cells to the Acutely Infarcted Myocardium

Objectives: Intracoronary cell transplantation during catheter balloon inflations may be associated with adverse events. We studied the effectiveness of an alternative transplantation technique – intracoronary cell infusion. Methods: Fourteen pigs, which had survived acute myocardial infarction, were randomized into 2 treatment groups and 2 controls. Three days after infarction, 12 pigs underwent allogeneic intracoronary mononuclear bone marrow cell transplantation using either the standard technique (short-term cell injections during repeat balloon inflations, technique A, n = 6) or continuous intracoronary cell infusion without balloon inflations (technique B, n = 6). Implanted cells were stained with fluorescent dye. After transplantation, the pigs were euthanized and myocardial samples were analyzed by fluorescent microscopy. Results: The mean numbers of fluorescently labeled bone marrow cells in the infarction border zone, in the infarction mid-area and in the center of myocardial infarction were 84, 72 and 55 using technique A, and 29, 57 and 46 using technique B, respectively. The mean cell retention in the infarction border zone of 84 cells for technique A and 29 cells for technique B differed significantly (p = 0.034, two-tailed t test). Conclusion: The continuous intracoronary cell infusion technique is a less efficient cell delivery technique as compared with the standard technique using repeat intracoronary balloon inflations.

Detecting minimal residual disease in patients with chronic lymphocytic leukemia using 8-color flow cytometry protocol in routine hematological practice.

Minimal residual disease (MRD) detection has become increasingly important for the assessment of therapy response in chronic lymphocytic leukemia (CLL). However, current MRD analysis methods, both molecular genetic and flow cytometric, are time‐consuming and require experienced laboratory staff.

Trypanosomatid mitochondrial RNA editing: Dramatically complex transcript repertoires revealed with a dedicated mapping tool

Abstract RNA editing by targeted insertion and deletion of uridine is crucial to generate translatable mRNAs from the cryptogenes of the mitochondrial genome of kinetoplastids. This type of editing consists of a stepwise cascade of reactions generally proceeding from 3′ to 5′ on a transcript, resulting in a population of partially edited as well as pre-edited and completely edited molecules for each mitochondrial cryptogene of these protozoans. Often, the number of uridines inserted and deleted exceed the number of nucleotides that are genome-encoded. Thus, analysis of kinetoplastid mitochondrial transcriptomes has proven frustratingly complex. Here we present our analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, our new tool which allows comprehensive characterization of RNA editing, not relying on targeted transcript amplification and on prior knowledge of final edited products. T-Aligner implements a pipeline of read mapping, visualization of all editing states and their coverage, and assembly of canonical and alternative translatable mRNAs. We also assess T-Aligner functionality on a more challenging deep sequencing read input from Trypanosoma cruzi. The analysis reveals that transcripts of cryptogenes of both species undergo very complex editing that includes the formation of alternative open reading frames and whole categories of truncated editing products.

Specific p53 mutations do not impact results of alemtuzumab therapy among patients with chronic lymphocytic leukemia

A poor prognosis in chronic lymphocytic leukemia (CLL) is associated particularly with the presence of del(17p) aff ecting tumor suppressor gene TP53 . Th is deletion is in almost all cases of progressive leukemia accompanied by a mutation in the other TP53 allele, and in a smaller proportion of patients the TP53 mutation occurs also independently of del(17p) [1]. Recently, we demonstrated that patients with CLL harboring a missense mutation located in the p53 DNA-binding motifs (DBMs) (structurally well-defi ned parts of the DNA-binding domain) manifested a clearly shorter median survival in comparison with those having a missense mutation outside DBMs or a non-missense alteration [2]. However, a limitation of this study resulted from the unpredictable survival impact of diverse therapy given to patients with p53 mutations. Th e therapeutic approach currently taken for patients with CLL with loss and/or mutation of TP53 relies mostly on the use of agents which do not act through DNA damage followed by apoptosis induction. Th e success of this approach has been documented for the monoclonal antibody alemtuzumab. Monotherapy with alemtuzumab is now being recommended as fi rst-line therapy for patients with CLL with del(17p) [3]; however, many unresolved questions need to be addressed. For example, it is unclear whether the type of p53 mutation infl uences the outcome of alemtuzumab therapy. In an eff ort to evaluate the effi cacy of alemtuzumab therapy in relation to specifi c p53 mutations in patients with CLL, we performed a retrospective analysis. Th e patients examined ( n 111) were treated by alemtuzumab between 2003 and 2010. Th e majority of cases concerned pretreated patients ( n 108; see Table I). Our present analysis was performed in the patient cohort which was largely included in the previous survival analysis related to p53 mutation types; this study also involved patients treated with other drugs in addition to alemtuzumab [2] (overlap 88% in group 1; 82% in group 2; 0% in group 3; the groups are defi ned further below). Defects in p53 were assessed by the yeast functional assay coupled to sequencing of DNA templates from mutated yeast colonies. Th e remaining TP53 allele was analyzed using

Multiple productive IGH rearrangements denote oligoclonality even in immunophenotypically monoclonal CLL

Multiple productive IGH rearrangements denote oligoclonality even in immunophenotypically monoclonal CLL