Flow alteration
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- MeSH
- cerebrovaskulární poruchy MeSH
- mozkový krevní oběh analýza MeSH
- senioři MeSH
- Check Tag
- senioři MeSH
Epilepsy is one of the most common neurologic disorders affecting a substantial part of the population worldwide. Epileptic seizures represent the situation of increased neuronal activity associated with the enhanced demands for sufficient energy supply. For that purpose, very efficient regulatory mechanisms have to operate to ensure that cerebral blood flow, delivery of oxygen, and nutrients are continuously adapted to the local metabolic needs. The sophisticated regulation has to function in concert at several levels (systemic, tissue, cellular, and subcellular). Particularly, mitochondria play a key role not only in the energy production, but they are also central to many other processes including those leading to neuronal death. Impairment of any of the involved pathways can result in serious functional alterations, neurodegeneration, and potentially in epileptogenesis. The present review will address some of the important issues concerning vascular and metabolic changes in pathophysiology of epilepsy.
- MeSH
- epilepsie komplikace genetika MeSH
- lidé MeSH
- metabolické nemoci etiologie genetika MeSH
- mitochondriální DNA genetika MeSH
- mozek metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.
Lipid bilayer properties are quantified with a variety of arbitrary selected parameters such as molecular packing and dynamics, electrostatic potentials or permeability. In the paper we determined the effect of phloretin and 6-ketocholestanol (dipole potential modifying agents) on the membrane hydration and efficiency of the trans-membrane water flow. The dynamics of water molecules within the lipid bilayer interface was evaluated using solvent relaxation method, whereas the osmotically induced trans-membrane water flux was estimated with the stopped-flow method using the liposome shrinkage kinetics. The presence of phloretin or 6-ketocholestanol resulted in a change of both, the interfacial hydration level and osmotically driven water fluxes. Specifically, the presence of 6-ketocholestanol reduced the amount and mobility of water in the membrane interface. It also slows the osmotically induced water flow. The interfacial hydration change caused by phloretin was much smaller and the effect on osmotically induced water flow was opposite to that of 6-ketocholestanol.
- MeSH
- floretin chemie MeSH
- fluorescenční barviva chemie MeSH
- ketocholesteroly chemie MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- liposomy chemie metabolismus MeSH
- permeabilita MeSH
- simulace molekulární dynamiky * MeSH
- voda chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Introduction: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVID patients. Methods: In this collaborative multicentric study the EuroFlow PID 8-color Pre-GC B-cell tube, standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD). Results: The Pre-GC B-cell tube allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5+ CD38+/++ CD21het CD24++ (2.7 vs 5.6 cells/µl, p=0.0004) and CD5+ CD38het CD21+ CD24+ (6.5 vs 17 cells/µl, p<0.0001) immature B cells (below normal HD levels in 22% and 37% of CVID patients). This was associated with an expansion of CD21-CD24- (6.1 vs 0.74 cells/µl, p<0.0001) and CD21-CD24++ (1.8 vs 0.4 cells/µl, p<0.0001) naïve B-cell counts above normal values in 73% and 94% cases, respectively. Additionally, reduced IgMD+ (21 vs 32 cells/µl, p=0.03) and IgMD- (4 vs 35 cells/µl, p<0.0001) MBC counts were found to be below normal values in 25% and 77% of CVID patients, respectively, always together with severely reduced/undetectable circulating blood pb. Comparison of the maturation pathway profile of pre-GC B cells in blood of CVID patients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%). Conclusion: Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID.
- MeSH
- běžná variabilní imunodeficience diagnóza imunologie metabolismus MeSH
- CD antigeny analýza MeSH
- dospělí MeSH
- fenotyp MeSH
- imunofenotypizace * MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- prekurzorové B-lymfoidní buňky imunologie metabolismus MeSH
- průtoková cytometrie * MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- studie případů a kontrol MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
The distribution of fluorescence signals measured with flow cytometry can be influenced by several factors, including qualitative and quantitative properties of the used fluorochromes, optical properties of the detection system, as well as the variability within the analyzed cell population itself. Most of the single cell samples prepared from in vitrocultures or clinical specimens contain a variable cell cycle component. Cell cycle, together with changes in the cell size, are two of the factors that alter the functional properties of analyzed cells and thus affect the interpretation of obtained results. Here, we describe the association between cell cycle status and cell size, and the variability in the distribution of fluorescence intensity as determined with flow cytometry, at population scale. We show that variability in the distribution of background and specific fluorescence signals is related to the cell cycle state of the selected population, with the 10% low fluorescence signal fraction enriched mainly in cells in their G0/G1 cell cycle phase, and the 10% high fraction containing cells mostly in the G2/M phase. Therefore we advise using caution and additional experimental validation when comparing populations defined by fractions at both ends of fluorescence signal distribution to avoid biases caused by the effect of cell cycle and cell size.
- MeSH
- buněčné dělení MeSH
- buněčný cyklus fyziologie MeSH
- G2 fáze * MeSH
- průtoková cytometrie metody MeSH
- velikost buňky MeSH
- Publikační typ
- časopisecké články MeSH
AIMS: This study compared the results obtained by basic immunophenotyping of cerebrospinal fluid (CSF) cells by flow cytometry (FC) to the results of conventional cytology and evaluated the possibility of detailed analyses of CSF B-cell subpopulations. METHODS: Samples from 42 patients were examined by conventional cytology (native and/or pre-centrifuged CSF) and FC. The results from 15 patients without evidence of organic neurological disease were used to estimate reference ranges. RESULTS: Pre-centrifugated CSF had significantly higher cell yield on the cytologic slide, but cell subpopulation percentages were altered; the percentage of lymphocytes was significantly higher and monocytes significantly lower compared to both native CSF slides and FC. The percentage of granulocytes was higher in FC compared to cytology. For leukocyte count, the following reference ranges were estimated for Fuchs-Rosenthal chamber (FR) counting and FC, respectively: leukocytes ≤4.7/μL and ≤2.5/μL, lymphocytes ≤4.1/μL and ≤1.8/μL, monocytes ≤1.2/μL and ≤0.9/μL, and granulocytes 0/μL and ≤0.2/μL. The following reference ranges were estimated for basic subpopulations: T-lymphocytes 84.1-100%, B lymphocytes 0.0-1.5%, NK cells 0.0-6.3%, NKT cells 0-9.5%, and CD3+CD4+/CD3+CD8+ 0.8-4.9. Using a volume of 1.2-2.4 mL, the number of B lymphocytes was too low (<20) in samples with ≤2.7 cells/μL in the FR. CONCLUSIONS: Even normal CSF samples are amenable to basic mononuclear cell subpopulation analysis by FC. However, analysis of the B-cell subpopulations requires either a larger sample volume or selection of samples with ≥ 3 cells/μL.
- MeSH
- imunofenotypizace MeSH
- leukocyty * MeSH
- lidé MeSH
- lymfocyty * MeSH
- mozkomíšní mok MeSH
- průtoková cytometrie metody MeSH
- T-lymfocyty MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Recently, several studies have demonstrated a negative prognostic impact of Ikaros (IKZF1) gene alterations in acute lymphoblastic leukemia (ALL). However, controversies still exist regarding the impact of IKZF1 in current treatment protocols. PROCEDURE: We simultaneously detected IKZF1 gene deletions by multiplex ligation-dependent probe amplification and gene expression of IKZF1 isoforms in 206 children with BCR/ABL-negative ALL treated with ALL IC-BFM 2002 protocol, in which risk stratification was not based on minimal residual disease (MRD), and validated the results on a cohort of 189 patients treated with MRD-directed ALL-BFM 2000 protocol. RESULTS: Deletion of IKZF1 was present in 14 of 206 (7%) ALL IC patients. Interestingly, gene expression did not completely correlate with the deletion status in either cohort. Deletions were not always reflected in the gene expression of dominant-negative isoforms, and conversely, 7 of 395 (2%) non-deleted cases overexpressed dominant-negative isoform Ik6. IKZF1 deletions significantly affected event-free survival (EFS) of the ALL IC cohort (41 ± 14% vs. 86 ± 3%, P < 0.0001). Regarding IKZF1 isoforms, only Ik6 overexpression had negative prognostic impact (EFS 50 ± 16% vs. 85 ± 3%, P = 0.003). In multivariate analysis, which included ALL IC risk criteria, flow-cytometric MRD and IKZF1 alterations, day 15 MRD and IKZF1 deletion status displayed an independent prognostic impact. CONCLUSIONS: We show that MRD-directed treatment diminishes prognostic impact of IKZF1 alterations. However, IKZF1 status alone or combined with day 15 flow cytometry can significantly improve risk stratification within BFM protocols at centers that do not perform antigen-receptor-based MRD monitoring.
- MeSH
- akutní lymfatická leukemie genetika mortalita patologie MeSH
- bcr-abl fúzní proteiny genetika MeSH
- dítě MeSH
- lidé MeSH
- předškolní dítě MeSH
- přežití bez známek nemoci MeSH
- prognóza MeSH
- průtoková cytometrie MeSH
- reziduální nádor genetika patologie MeSH
- rizikové faktory MeSH
- transkripční faktor Ikaros genetika MeSH
- transkriptom MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
