The present nuclear and cell body diameter measurements demonstrated size differences of the approximate cell space estimate occupied by the cell nucleus during the cell differentiation in lymphocytic, granulocytic and erythroid cell lineages. These lineages were used as convenient models because all differentiation steps were easily identified and accessible in diagnostic peripheral blood or bone marrow smears of blood donors (BDs), patients suffering from chronic lymphocytic leukemia (CLL), patients with chronic myeloid leukemia (CML) and refractory anemia (RA) of the myelodysplastic syndrome (MDS). The cell space occupied by the nucleus was constant and did not change during the cell differentiation in the lymphocytic cell lineages of BDs and CLL patients despite the decreased cell size. In contrary, the cell space occupied by the nucleus markedly decreased in differentiating cells of granulocytic and erythroid lineages of patients suffering from CML. In the erythroid cell lineage in patients with RA of MDS the small reduction of the cell space occupied by the nucleus during the differentiation was not significant. The measurements also indicated that in progenitor cells of all studied cell lineages nuclei occupied more than 70 % of the cell space. Thus, the nucleus-cytoplasmic morphological and functional equilibrium appeared to be characteristic for each differentiation step and each specific cell lineage.
- MeSH
- buněčná diferenciace * MeSH
- buněčné jádro * MeSH
- chronická lymfatická leukemie patologie MeSH
- chronická myeloidní leukemie patologie MeSH
- erytroidní buňky cytologie MeSH
- granulocyty cytologie MeSH
- lidé MeSH
- lymfocyty cytologie MeSH
- refrakterní anemie patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Hematopoiesis, the formation of blood cells from hematopoietic stem cells (HSC), is a highly regulated process. Since the discovery of microRNAs (miRNAs), several studies have shown their significant role in the regulation of the hematopoietic system. Impaired expression of miRNAs leads to disrupted cellular pathways and in particular causes loss of hematopoietic ability. Here, we report a previously unrecognized function of miR-143 in granulopoiesis. Hematopoietic cells undergoing granulocytic differentiation exhibited increased miR-143 expression. Overexpression or ablation of miR-143 expression resulted in accelerated granulocytic differentiation or block of differentiation, respectively. The absence of miR-143 in mice resulted in a reduced number of mature granulocytes in blood and bone marrow. Additionally, we observed an association of high miR-143 expression levels with a higher probability of survival in two different cohorts of patients with acute myeloid leukemia (AML). Overexpression of miR-143 in AML cells impaired cell growth, partially induced differentiation, and caused apoptosis. Argonaute2-RNA-Immunoprecipitation assay revealed ERK5, a member of the MAPK-family, as a target of miR-143 in myeloid cells. Further, we observed an inverse correlation of miR-143 and ERK5 in primary AML patient samples, and in CD34+ HSPCs undergoing granulocytic differentiation and we confirmed functional relevance of ERK5 in myeloid cells. In conclusion, our data describe miR-143 as a relevant factor in granulocyte differentiation, whose expression may be useful as a prognostic and therapeutic factor in AML therapy.
- MeSH
- 3' nepřekládaná oblast MeSH
- akutní myeloidní leukemie metabolismus mortalita patologie MeSH
- antagomiry metabolismus MeSH
- apoptóza MeSH
- buněčná diferenciace MeSH
- granulocyty cytologie metabolismus MeSH
- hematopoetické kmenové buňky cytologie metabolismus MeSH
- lidé MeSH
- mikro RNA antagonisté a inhibitory genetika metabolismus MeSH
- míra přežití MeSH
- mitogenem aktivovaná proteinkinasa 7 chemie genetika metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- prognóza MeSH
- proliferace buněk MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- buněčný rodokmen MeSH
- granulocyty cytologie MeSH
- hematopoéza * MeSH
- myši knockoutované MeSH
- myši MeSH
- proteiny vázající zesilovač transkripce CCAAT nedostatek fyziologie MeSH
- transkripční faktory MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.
- MeSH
- akutní myeloidní leukemie metabolismus patologie MeSH
- apoptóza MeSH
- buněčná diferenciace účinky léků MeSH
- buňky kostní dřeně cytologie MeSH
- down regulace účinky léků MeSH
- estradiol farmakologie MeSH
- faktor stimulující kolonie granulocytů farmakologie MeSH
- granulocyty cytologie metabolismus MeSH
- hematopoetické kmenové buňky cytologie metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- membránové glykoproteiny antagonisté a inhibitory genetika metabolismus MeSH
- membránové proteiny antagonisté a inhibitory genetika metabolismus MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- proliferace buněk účinky léků MeSH
- promotorové oblasti (genetika) MeSH
- protein alfa vázající zesilovač transkripce CCAAT genetika metabolismus MeSH
- RNA interference MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Alu-mediated tandem duplication of exons 4 and 5 (g.15815_22218dup6404) is a novel mutation that has been detected in the LAMP2 gene (Xq24). This exon copy number variation was found in two brothers with the typical phenotype of Danon disease, including characteristic myocardial changes on magnetic resonance imaging. The 6.4 kb duplication was identified in both boys by a combination of exon dosage qPCR analyses and duplication breakpoint/junction mapping. The rearrangement results in a plethora of abnormal LAMP2 splicing variants and also in use of likely cryptic splice sites in the 3' terminus of LAMP2 gene. Although we found minute amounts of normal LAMP2B and LAMP2A mRNAs, no protein was detectable in peripheral blood leukocytes by flow cytometry in both brothers. Uniquely, the fraction of LAMP2-deficient granulocytes (0.06%) assessed by flow cytometry in the patients' asymptomatic mother substantially differed from the random distribution of X-chromosome inactivation in her leukocytes. This discrepancy was later explained by molecular genetic methods as a consequence of mosaic distribution of the mutation in her somatic tissues. Altogether, we report a novel and mosaically distributed exon copy number rearrangement in the LAMP2 gene and comment on obstacles this genetic setup presents to the overall cellular and molecular diagnostic algorithm of Danon disease. Our observations of the mosaicism in the asymptomatic mother suggest that similarly affected females could be a potentially under-diagnosed Danon disease carrier group and that LAMP2 flow cytometry, because of its supreme sensitivity, can be an efficient method for pedigree screening.
- MeSH
- dospělí MeSH
- duplikace genu * MeSH
- exony * MeSH
- fenotyp MeSH
- glykogenóza typu IIb diagnóza genetika MeSH
- granulocyty cytologie MeSH
- leukocyty cytologie MeSH
- lidé MeSH
- magnetická rezonanční tomografie MeSH
- membránový protein 2 asociovaný s lyzozomy genetika MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mozaicismus MeSH
- mutace MeSH
- myokard patologie MeSH
- průtoková cytometrie MeSH
- rodokmen MeSH
- sourozenci MeSH
- tkáňová distribuce MeSH
- variabilita počtu kopií segmentů DNA MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
Inflammatory processes play an important role in the development of nasal polyps (NP), but the etiology and, to a high degree also, the pathogenesis of NP are not fully understood. The role of several cytokines and chemokines such as eotaxins, IL-4, IL-5, IL-6, IL-8, and RANTES has been reported in NP. Herewith, we investigated the expression and pattern of distribution of chemokine receptors CCR1 and CCR3 in nasal polyps. Immunohistochemical detection was carried out in frozen sections of biopsies from 22 NP and 18 nasal mucosa specimens in both the epithelial and stromal compartments. Fluorescence microscopy and computerized image analysis revealed a statistically significant increased number of CCR1 (45.2 ± 2.8 vs. 15.1 ± 1.9, p < 0.001)-positive as well as CCR3 (16.4 ± 1.4 vs. 9.7 ± 1.1, p < 0.001)-positive cells in the stroma of NP compared to nasal mucosa. In comparison to healthy nasal mucosa, increased positivity of CCR3 was detected in the epithelial compartment of NP. Our data suggest that increased expression of CCR1 and CCR3 chemokine receptors may, in accord with various chemokines, contribute to the pathogenesis of nasal polyposis by facilitating increased migration and prolonged accumulation of inflammatory cells, e.g., eosinophils, in the inflammatory infiltrate of NP.
- MeSH
- eozinofily cytologie MeSH
- granulocyty cytologie MeSH
- lidé MeSH
- nosní polypy genetika metabolismus patologie MeSH
- nosní sliznice metabolismus patologie MeSH
- receptory CCR1 genetika metabolismus MeSH
- receptory CCR3 genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The present study was undertaken to provide complementary data on the heterochromatin condensation in both central and peripheral nuclear regions during the cell differentiation and maturation using computer-assisted density measurements at the single-cell level. The lineage of neutrophilic granulocytes in the bone marrow of patients suffering from chronic myeloid leukaemia was very convenient for such study because the increased number of granulocytes in all developmental stages was satisfactory for heterochromatin density measurements. The morphology of leukaemic and non-leukaemic neutrophilic granulocytes is similar and each differentiation or maturation stage is easily identified. A markedly increasing heterochromatin density--condensation--in the peripheral nuclear region at the nuclear envelope accompanied both the differentiation and maturation of these cells. Thus, peripheral chromosomal territories at the nuclear envelope are important for both the differentiation and maturation process. In contrast, the heterochromatin density of nuclear central regions was already high in early differentiation stages and exhibited a less distinct increase during the differentiation, but was more apparent in late maturation stages representing the terminal differentiation. A limited number of maturing cells with persisting large heterochromatin density in central nuclear regions without markedly increased heterochromatin condensation at the nuclear periphery might represent a further maturation abnormality--asynchrony--during the granulocytic development. From the methodological point of view, both, the cytochemical method for the DNA demonstration and the panoptic May-Grünwald-Giemsa staining, are convenient for computer-assisted chromatin densitometry at the single-cell level.
- MeSH
- buněčná diferenciace MeSH
- buněčné jádro metabolismus MeSH
- buněčný rodokmen MeSH
- chromatin chemie metabolismus MeSH
- chronická myeloidní leukemie metabolismus MeSH
- DNA chemie MeSH
- granulocyty cytologie metabolismus MeSH
- heterochromatin metabolismus MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
In this study we examined differences in selected indices of granulopoiesis in outbred, F(1) hybrid and inbred mouse strains. Specifically, serum granulocyte colony-stimulating factor (G-CSF) levels, numbers of marrow granulocyte-macrophage progenitor cells and morphologically recognizable proliferative marrow granulocytic precursor cells were evaluated. These parameters were determined in untreated controls, and in mice exposed either to a non-specific stimulus (injection of saline) or to a granulopoiesis-enhancing stimulus (administration of a cyclooxygenase-2 inhibitor, meloxicam). Lower levels of G-CSF were detectable in the outbred ICR mice, which also demonstrated an enhanced response to both types of the stimuli. Considering the fact that outbred mice are closer to natural mammalian populations, including human ones, the possibility of using outbred mice, instead of the often used inbred strains, for experiments evaluating the effects of pharmacological interventions on hematopoiesis should be investigated.
- MeSH
- buňky kostní dřeně cytologie účinky léků MeSH
- chlorid sodný aplikace a dávkování farmakologie MeSH
- faktor stimulující granulocyto-makrofágové kolonie krev MeSH
- faktor stimulující kolonie granulocytů krev MeSH
- granulocyty cytologie účinky léků MeSH
- hybridizace genetická genetika MeSH
- inbrední kmeny myší MeSH
- inbreeding MeSH
- inhibitory cyklooxygenasy farmakologie MeSH
- injekce intraperitoneální MeSH
- lidé MeSH
- myelopoéza účinky léků fyziologie MeSH
- myši MeSH
- progenitory granulocytů a makrofágů MeSH
- proliferace buněk účinky léků MeSH
- thiaziny farmakologie MeSH
- thiazoly farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Isolation of granulocytes from blood is necessary for accurate study of changes in their expression. After gradient centrifugation, we obtain relatively pure granulocyte populations with different ratios of neutrophils and eosinophils. Unfortunately, in many studies in this field the expression results are not set according to the real variability of the granulocyte population. In many cases, the granulocyte population is marked simply as "neutrophils" and the residual population of eosinophils is not considered. Based on our recent study where we tracked the general transcription factor RNA polymerase II, we hypothesized that eosinophils are more transcriptionally active cells than neutrophils. We decided to test our hypothesis on isolated cells because its implications could change our view on many past expression analyses performed on granulocytes. In our experiments, we isolated neutrophils and eosinophils and measured their total RNA production. According to our results, eosinophils produce much more RNA than neutrophils. Therefore, relatively low numbers of highly active eosinophils can markedly affect the whole pool of granulocytic RNA. We want to emphasize that either a detailed description of the cell population or the use of a pure neutrophil population is necessary for the correct interpretation of neutrophil expression analysis results.
- MeSH
- cytologické techniky metody MeSH
- granulocyty cytologie patologie MeSH
- intrakraniální krvácení MeSH
- lidé MeSH
- lymfocyty cytologie patologie MeSH
- mononukleární fagocytární systém cytologie patologie MeSH
- mozkomíšní mok cytologie chemie mikrobiologie MeSH
- nádorové cirkulující buňky klasifikace patologie MeSH
- patologické procesy etiologie klasifikace patologie MeSH
- Check Tag
- lidé MeSH
