Flow disruption Dotaz Zobrazit nápovědu
We have recently demonstrated that the alkaloid colchicine (COL) inhibits glucocorticoid receptor (GR) transcriptional activity. In addition, we described proteasome-mediated degradation of GR in COL-treated HeLa cells. While these effects were previously attributed to cell cycle arrest in G2/M phase, this explanation is not applicable for nonproliferating cells such as human hepatocytes (HH). In the current study, we compared COL-mediated microtubule disruption and cell cycle arrest with selected GR functions in HeLa cells and HH as models of proliferating and quiescent cells, respectively. Microtubule disruption led to irreversible decrease in GR binding capacity and protein level in HeLa cells. None of the parameters was restored 24 hours after COL withdrawal. In contrast, dexamethasone (DEX) binding was increased in HH at the beginning of the treatment, with following transient activation of extracellular signal-regulated kinase (ERK). The findings of these investigations emphasize the GR-signaling differences between primary and transformed cells.
- MeSH
- aktivace enzymů MeSH
- buněčný cyklus účinky léků MeSH
- časové faktory MeSH
- dexamethason metabolismus MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- fluorescenční mikroskopie MeSH
- HeLa buňky MeSH
- hepatocyty účinky léků metabolismus ultrastruktura MeSH
- kolchicin farmakologie MeSH
- lidé MeSH
- ligandy MeSH
- mikrotubuly účinky léků ultrastruktura MeSH
- modulátory tubulinu farmakologie MeSH
- proliferace buněk účinky léků MeSH
- průtoková cytometrie MeSH
- receptory glukokortikoidů metabolismus fyziologie MeSH
- signální transdukce účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- dexamethason MeSH
- dexamethasone receptor MeSH Prohlížeč
- extracelulárním signálem regulované MAP kinasy MeSH
- kolchicin MeSH
- ligandy MeSH
- modulátory tubulinu MeSH
- receptory glukokortikoidů MeSH
Several methods were tested that would improve the fluorescence signal from hybridized rumen bacterial cells. Disruption of cell envelopes by lysozyme, EDTA, proteinase K and/or SDS caused only a minor increase in fluorescence signal. Use of helper unlabeled oligonucleotide probes was successful only with the Puni[H672] probe which, however, when used with specific PBBl4-labeled probe, gave fluorescence signal drop. No substantial rise in fluorescence signal was also observed with cells subjected to growth-without-cell-division treatment. Further improvements are needed to make the fluorescent in situ hybridization (FISH)-flow cytometry combination applicable to rumen bacteria.
- MeSH
- bachor mikrobiologie MeSH
- Bacteria genetika izolace a purifikace MeSH
- bakteriologické techniky * MeSH
- barvení a značení MeSH
- DNA sondy MeSH
- fluorescenční barviva MeSH
- hybridizace in situ fluorescenční MeSH
- průtoková cytometrie * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA sondy MeSH
- fluorescenční barviva MeSH
INTRODUCTION: 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. METHODS: To reduce the demands of flow cytometric MRD detection in CLL, we have introduced a novel flow cytometric 8-color protocol. The MRD analysis results using this protocol were then compared with the commonly employed 4-color protocol and the molecular genetic (real-time quantitative allele-specific oligonucleotide IGH polymerase chain reaction; RQ-ASO IGH PCR) approach. RESULTS: Forty-two CLL patient samples were repeatedly analyzed after allogeneic stem cell transplantation (n = 20) or after fludarabine-based therapy (n = 22), and 100% concordance was found using both flow cytometric protocols. Furthermore, there was a strong correlation (r = 0.94) between flow cytometric and RQ-ASO IGH PCR results in MRD detection. CONCLUSION: Flow cytometry is less time-consuming, less financially demanding, and moreover, MRD assessment using our novel 8-color protocol is less complicated than the 4-color approach and molecular methods.
- Klíčová slova
- Chronic lymphocytic leukemia, flow cytometry, minimal residual disease,
- MeSH
- chronická lymfatická leukemie krev patologie terapie MeSH
- dospělí MeSH
- homologní transplantace MeSH
- imunofenotypizace metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- průtoková cytometrie metody MeSH
- reprodukovatelnost výsledků MeSH
- reziduální nádor diagnóza MeSH
- senioři MeSH
- senzitivita a specificita MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- transplantace hematopoetických kmenových buněk MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- těžké řetězce imunoglobulinů MeSH
The major risk factor for Clostridium difficile infection (CDI) is the use of antibiotics owing to the disruption of the equilibrium of the host gut microbiota. To preserve the beneficial resident probiotic bacteria during infection treatment, the use of molecules with selective antibacterial activity enhances the efficacy by selectively removing C. difficile. One of them is the plant alkaloid 8-hydroxyquinoline (8HQ), which has been shown to selectively inhibit clostridia without repressing bifidobacteria. Selective antimicrobial activity is generally tested by culture techniques of individual bacterial strains. However, the main limitation of these techniques is the inability to describe differential growth dynamics of more bacterial strains in co-culture within the same experiment. In the present study, we combined fluorescent in situ hybridization and flow cytometry to describe the changes in active and non-active cells of a mixed culture formed by the opportunistic pathogen C. difficile CECT 531 and the beneficial Bifidobacterium longum subsp. longum CCMDMND BL1 after exposure to 8HQ. It was observed that without 8HQ, the proportion of both strains was almost equal, oscillating between 22.7 and 77.9 % during a time lapse of 12 h, whereas with 8HQ the proportion of active C. difficile decreased after 4 h, and persisted only between 8.8 and 17.5 %. In contrast, bifidobacterial growth was not disturbed by 8HQ. The results of this study showed the selective inhibitory effect of 8HQ on clostridial and bifidobacterial growth dynamics, and the potential of this compound for the development of selective agents to control CDIs.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bifidobacterium účinky léků růst a vývoj MeSH
- Clostridioides difficile účinky léků růst a vývoj MeSH
- hybridizace in situ fluorescenční MeSH
- mikrobiální interakce MeSH
- oxychinolin farmakologie MeSH
- průtoková cytometrie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- oxychinolin MeSH
The mechanism of colloidal silica action to improve flow properties of pharmaceutical powders is known to be based on inter-particle force disruption by silica particles adhered to the particle surface. In the present article, the kinetic aspects of this action are investigated, focusing on non-spherical particles of different size. Blends comprising microcrystalline cellulose or calcium hydrogen phosphate dihydrate and colloidal silica were examined using powder rheometer. The blends were formulated to represent effects of particle size, surface texture, colloidal silica loading, and mixing time. Pre-conditioning, shear testing, compressibility, and flow energy measurements were used to monitor flow properties. Components and blends were analyzed using particle size analysis and scanning electron microscopy (SEM), using energy dispersive spectroscopy (EDS) and back-scattered electron (BSE) detection to determine surface particle arrangement. All studied parameters were found to have substantial effects on flow properties of powder blends. Those effects were explained by identifying key steps of colloidal silica action, which were found to proceed at substantially different rates, causing the flow properties change over time being dependent on the blend formulation and the component properties.
- Klíčová slova
- Colloidal silica, Flow properties, Flow-enhancer, Glidant, Powder mixing, Powder rheology,
- MeSH
- časové faktory MeSH
- celulosa chemie MeSH
- chemie farmaceutická metody MeSH
- fosforečnany vápenaté chemie MeSH
- koloidy chemie MeSH
- mikroskopie elektronová rastrovací MeSH
- oxid křemičitý chemie MeSH
- pomocné látky chemie MeSH
- prášky, zásypy, pudry MeSH
- příprava léků metody MeSH
- reologie MeSH
- spektrometrie rentgenová emisní MeSH
- velikost částic MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- calcium phosphate, dibasic, dihydrate MeSH Prohlížeč
- celulosa MeSH
- fosforečnany vápenaté MeSH
- koloidy MeSH
- microcrystalline cellulose MeSH Prohlížeč
- oxid křemičitý MeSH
- pomocné látky MeSH
- prášky, zásypy, pudry MeSH
The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as "required" or "recommended" for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate "required" markers using normal peripheral blood was developed. Responses were received from 150 participants with a diagnostic workload >20 CLL cases per week in 23/150 (15%), 5-20 in 82/150 (55%), and <5 cases per week in 45/150 (30%). The consensus for "required" diagnostic markers included: CD19, CD5, CD20, CD23, Kappa, and Lambda. "Recommended" markers potentially useful for differential diagnosis were: CD43, CD79b, CD81, CD200, CD10, and ROR1. Reproducible criteria for component reagents were assessed retrospectively in 14,643 cases from 13 different centers and showed >97% concordance with current approaches. A pilot study to validate staining quality was completed in 11 centers. Markers considered as "required" for the diagnosis of CLL by the participants in this study (CD19, CD5, CD20, CD23, Kappa, and Lambda) are consistent with current diagnostic criteria and practice. Importantly, a reproducible approach to validate and apply these markers in individual laboratories has been identified. Finally, a consensus "recommended" panel of markers to refine diagnosis in borderline cases (CD43, CD79b, CD81, CD200, CD10, and ROR1) has been defined and will be prospectively evaluated. © 2017 International Clinical Cytometry Society.
- Klíčová slova
- chronic lymphocytic leukemia, diagnosis, flow cytometry,
- MeSH
- chronická lymfatická leukemie diagnóza metabolismus MeSH
- imunofenotypizace metody MeSH
- lidé MeSH
- nádorové biomarkery metabolismus MeSH
- pilotní projekty MeSH
- průtoková cytometrie metody MeSH
- reprodukovatelnost výsledků MeSH
- retrospektivní studie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- nádorové biomarkery MeSH
BACKGROUND: For examination of surface characteristics of lymphocytes automatic analyzers of flow cytometry are used which make it possible to assess labelling with 2-3 monoclonal antibodies on one examined cell and provide thus more accurate data on the immunophenotype of proliferating cells. The objective of the present work was-using the method of flow cytometry and monoclonal antibodies-to detect surface signs on lymphocytes in the peripheral blood stream of patients with lymphatic leukaemia. METHODS AND RESULTS: In 29 patients with chronic lymphatic leukaemia type B (B-CLL) CD signs were examined, using flow cytometry and the findings were compared with those obtained in a control group of 63 healthy subjects. Evidence of CD 19+ CD5+ lymphocytes (B1 cells) in B-CLL (83.4 +/- 15% vs. 3.5 +/- 1.9% in controls) was of diagnostic importance. These CD5+ B cells are polyreactive, "memory B" lymphocytes which contrary to "conventional" lymphocytes B2 (CD5-B) do not differentiate further to plasma cells. Accumulation of B1 (CD5+ B) lymphocytes was, on the other hand, associated in patients with B-CLL with a decrease of B2 (CD5-B) lymphocytes (2.8 +/- 4.0% vs 9.0 +/- 4.2%) (p < 0.01). This finding can in case of excess of B1 (CD5+ B) lymphocytes explain the tendency of patients with B-CLL to develop autoimmune complications (e.g. AIHA); and conversely a decrease of B2 (CD5-B) lymphocytes can lead to hypogammaglobulinaemia which is also associated with B-CLL. A tendency towards autoimmunity may be also promoted by a decline of so-called inductors of suppressor cells (CD 4+ CD45RA); in patients with B-CLL among CD4 T lymphocytes 32.5 +/- 15% CD 45RA+ cells were found, as compared to 43.0 +/- 14.8% in controls (p < 0.011) and a lower ratio of true suppressor T cells (CD8+ CD11b+) in patients with B-CLL: 47.0 +/- 17.6% CD11b+ from CD8+ cells, as compared with controls 80.4 +/- 13.3 CD11b from CD 8+ cells (p < 0.01). CONCLUSIONS: The method of flow cytometry with double fluorescence thus contributes to the accurate diagnosis of B-CLL, to monitoring of the therapeutic effect but also to knowledge of the immunopathology of the disease.
OBJECTIVES: A novel method of long-term telemetric monitoring of mean arterial pressure (MAP) and intracranial pressure (ICP) for the determination of current cerebral perfusion pressure (CPP) and the time course of ICP in freely moving rats under physiological conditions and with increased ICP due to the induced cerebral edema were studied. METHODS: The brain edema, that caused volume enlargement and ICP elevation was achieved in entirely experimental conditions without any parallel pathological process. Vasogenic/extracellular edema was induced by osmotic blood-brain barrier disruption (BBBd) and for induction of cytotoxic/intracellular edema the water intoxication model (WI) was used. RESULTS: The results showed significantly elevated values of ICP both in conditions of osmotic blood-brain barrier disruption (BBBd model) and cytotoxic/intracellular edema (WI model) compared to intact rats. The average values of ICP were significantly higher in WI model compared to osmotic BBBd model. Distinct pattern of elevated ICP, related to the selected way of experimental brain edema induction, was found. In the experimental model of osmotic BBB disruption, the elevation of ICP started earlier but was of very short duration. In WI model the elevation of ICP was present during the whole period of monitoring. CONCLUSION: Our results indicate that purely experimental models of brain edema (WI, BBBd) without any parallel pathological process can compromise the basic brain homeostatic activity.
- MeSH
- edém mozku diagnóza etiologie patofyziologie MeSH
- hematoencefalická bariéra patofyziologie MeSH
- intoxikace vodou komplikace diagnóza patofyziologie MeSH
- intrakraniální hypertenze komplikace diagnóza patofyziologie MeSH
- intrakraniální tlak fyziologie MeSH
- krysa rodu Rattus MeSH
- monitorování fyziologických funkcí metody MeSH
- mozek patofyziologie MeSH
- mozkový krevní oběh fyziologie MeSH
- potkani Wistar MeSH
- telemetrie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
ERGO (EndocRine Guideline Optimization) is the acronym of a European Union-funded research and innovation action, that aims to break down the wall between mammalian and non-mammalian vertebrate regulatory testing of endocrine disruptors (EDs), by identifying, developing and aligning thyroid-related biomarkers and endpoints (B/E) for the linkage of effects between vertebrate classes. To achieve this, an adverse outcome pathway (AOP) network covering various modes of thyroid hormone disruption (THD) in multiple vertebrate classes will be developed. The AOP development will be based on existing and new data from in vitro and in vivo experiments with fish, amphibians and mammals, using a battery of different THDs. This will provide the scientifically plausible and evidence-based foundation for the selection of B/E and assays in lower vertebrates, predictive of human health outcomes. These assays will be prioritized for validation at OECD (Organization for Economic Cooperation and Development) level. ERGO will re-think ED testing strategies from in silico methods to in vivo testing and develop, optimize and validate existing in vivo and early life-stage OECD guidelines, as well as new in vitro protocols for THD. This strategy will reduce requirements for animal testing by preventing duplication of testing in mammals and non-mammalian vertebrates and increase the screening capacity to enable more chemicals to be tested for ED properties.
- Klíčová slova
- AOP, IATA, OECD, adverse outcome pathway, biomarkers, cross-species extrapolation, endocrine disruption, integrated approach to testing and assessment, test guideline, thyroid hormone disruption,
- MeSH
- biologické markery MeSH
- biotest * metody MeSH
- druhová specificita MeSH
- endokrinní disruptory škodlivé účinky analýza MeSH
- endokrinní systém účinky léků metabolismus MeSH
- hodnocení rizik MeSH
- hodnocení vlivů na zdraví MeSH
- lidé MeSH
- monitorování životního prostředí * metody MeSH
- průběh práce MeSH
- skladování dat MeSH
- zdravotnické plány - realizace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- biologické markery MeSH
- endokrinní disruptory MeSH
In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.
- MeSH
- CD antigeny metabolismus MeSH
- chronická lymfatická leukemie patologie terapie MeSH
- dospělí MeSH
- imunofenotypizace MeSH
- kombinovaná terapie MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- následné studie MeSH
- prognóza MeSH
- průtoková cytometrie normy MeSH
- reziduální nádor diagnóza genetika metabolismus MeSH
- staging nádorů MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Názvy látek
- CD antigeny MeSH
