Bordetella pertussis infects human upper airways and deploys an array of immunosuppressive virulence factors, among which the adenylate cyclase toxin (CyaA) plays a prominent role in disarming host phagocytes. CyaA binds the complement receptor-3 (CR3 aka αMβ2 integrin CD11b/CD18 or Mac-1) of myeloid cells and delivers into their cytosol an adenylyl cyclase enzyme that hijacks cellular signaling through unregulated conversion of cytosolic ATP to cAMP. We found that the action of as little CyaA as 22 pM (4 ng/mL) blocks macrophage colony-stimulating factor (M-CSF)-driven transition of migratory human CD14+ monocytes into macrophages. Global transcriptional profiling (RNAseq) revealed that exposure of monocytes to 22 pM CyaA for 40 hours in culture with 20 ng/mL of M-CSF led to upregulation of genes that exert negative control of monocyte to macrophage differentiation (e.g., SERPINB2, DLL1, and CSNK1E). The sustained CyaA action yielded downregulation of numerous genes involved in processes crucial for host defense, such as myeloid cell differentiation, chemotaxis of inflammatory cells, antigen presentation, phagocytosis, and bactericidal activities. CyaA-elicited signaling also promoted deacetylation and trimethylation of lysines 9 and 27 of histone 3 (H3K9me3 and H3K27me3) and triggered the formation of transcriptionally repressive heterochromatin patches in the nuclei of CyaA-exposed monocytes. These effects were partly reversed by the G9a methyltransferase inhibitor UNC 0631 and by the pleiotropic HDAC inhibitor Trichostatin-A, revealing that CyaA-elicited epigenetic alterations mediate transcriptional reprogramming of monocytes and play a role in CyaA-triggered block of monocyte differentiation into bactericidal macrophage cells.IMPORTANCETo proliferate on host airway mucosa and evade elimination by patrolling sentinel cells, the whooping cough agent Bordetella pertussis produces a potently immunosubversive adenylate cyclase toxin (CyaA) that blocks opsonophagocytic killing of bacteria by phagocytes like neutrophils and macrophages. Indeed, chemotactic migration of CD14+ monocytes to the infection site and their transition into bactericidal macrophages, thus replenishing the exhausted mucosa-patrolling macrophages, represents one of the key mechanisms of innate immune defense to infection. We show that the cAMP signaling action of CyaA already at a very low toxin concentration triggers massive transcriptional reprogramming of monocytes that is accompanied by chromatin remodeling and epigenetic histone modifications, which block the transition of migratory monocytes into bactericidal macrophage cells. This reveals a novel layer of toxin action-mediated hijacking of functional differentiation of innate immune cells for the sake of mucosal pathogen proliferation and transmission to new hosts.

• Klíčová slova
• Bordetella pertussis, RTX toxins, cyclic AMP, differentiation, epigenetics, macrophages, monocytes,
• MeSH
• adenylátcyklasový toxin * metabolismus   MeSH
• Bordetella pertussis * patogenita   enzymologie   MeSH
• buněčná diferenciace * účinky léků   MeSH
• faktor stimulující kolonie makrofágů MeSH
• kultivované buňky MeSH
• lidé MeSH
• makrofágy * účinky léků   cytologie   MeSH
• monocyty * účinky léků   cytologie   fyziologie   MeSH
• přeprogramování buněk * MeSH
• restrukturace chromatinu * účinky léků   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• faktor stimulující kolonie makrofágů MeSH

OBJECTIVE: Blood monocyte subsets are emerging as biomarkers of cardiovascular inflammation. However, our understanding of human monocyte heterogeneity and their immunophenotypic features under healthy and inflammatory conditions is still evolving. RATIONALE: In this study, we sought to investigate the immunophenome of circulating human monocyte subsets. METHODS: Multiplexed, high-throughput flow cytometry screening arrays and computational data analysis were used to analyze the expression and hierarchical relationships of 242 specific surface markers on circulating classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes in healthy adults. RESULTS: Using generalized linear models and hierarchical cluster analysis, we selected and clustered epitopes that most reliably differentiate between monocyte subsets. We validated existing transcriptional profiling data and revealed potential new surface markers that uniquely define the classical (e.g., BLTR1, CD35, CD38, CD49e, CD89, CD96), intermediate (e.g., CD39, CD275, CD305, CDw328), and nonclassical (e.g., CD29, CD132) subsets. In addition, our analysis revealed phenotypic cell clusters, identified by dendritic markers CMRF-44 and CMRF-56, independent of the traditional monocyte classification. CONCLUSION: These results reveal an advancement of the clinically applicable multiplexed screening arrays that may facilitate monocyte subset characterization and cytometry-based biomarker selection in various inflammatory disorders.

• MeSH
• ateroskleróza diagnóza   imunologie   MeSH
• biodiverzita MeSH
• biologické markery metabolismus   MeSH
• fenotyp MeSH
• imunofenotypizace metody   MeSH
• krevní oběh MeSH
• lidé MeSH
• lipopolysacharidové receptory metabolismus   MeSH
• monocyty fyziologie   MeSH
• průtoková cytometrie MeSH
• receptory IgG metabolismus   MeSH
• rychlé screeningové testy MeSH
• separace buněk MeSH
• shluková analýza MeSH
• zánět diagnóza   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• lipopolysacharidové receptory MeSH
• receptory IgG MeSH

Circulating cell-free DNA (cfDNA) may be involved in immune response regulation. We studied the variations in abundance of telomeric sequences in plasma and serum in young healthy volunteers and the ability of cfDNA contained in these samples to co-activate the TNF-α m RNA expression in monocytes. We performed qPCR to determine relative telomere length (T/S ratios) in plasma, serum and whole blood of 36 volunteers. Using paired samples of plasma and serum and DNase treatment, we analysed the contribution of cfDNA to the co-activation of TNF-α mRNA expression in THP1 monocytic cell line. We found significant differences between paired plasma and serum samples in relative T/S ratios (median 1.38 ± 1.1 vs. 0.86 ± 0.25, respectively) and in total amounts of cfDNA and in estimated total amounts of telomeres which were significantly higher in serum than in plasma. TNF-α mRNA expression in THP1 cells increased significantly after DNase treatment of all samples used for stimulation. The highest TNF-α mRNA expressions were observed after stimulation with DNase treated serum samples. Our results suggest that the different content of telomeric sequences in plasma and serum may contribute to the tuning of immune response. Further studies of this interesting phenomenon are needed.

• MeSH
• deoxyribonukleasy metabolismus   MeSH
• homeostáza telomer MeSH
• imunita MeSH
• imunomodulace MeSH
• krevní plazma imunologie   metabolismus   MeSH
• lidé MeSH
• mladý dospělý MeSH
• monocyty fyziologie   MeSH
• sérum imunologie   metabolismus   MeSH
• telomery genetika   MeSH
• THP-1 buňky MeSH
• TNF-alfa genetika   metabolismus   MeSH
• upregulace MeSH
• volné cirkulující nukleové kyseliny genetika   imunologie   metabolismus   MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• Check Tag
• lidé MeSH
• mladý dospělý MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• deoxyribonukleasy MeSH
• TNF-alfa MeSH
• volné cirkulující nukleové kyseliny MeSH

Even though there is an abundance of information on the reference values of haematological parameters in adult rabbits, a little is known about the changes in haematology in newborn rabbits or during their postnatal development. Therefore, the aim of our study was to investigate changes in red blood cells (RBC), white blood cells (WBC) and differential leukocyte counts in SPF New Zealand White rabbits from the age of one day to 20 weeks. Significant age-related changes during the first four weeks of life were detected. These included an increase of RBC and WBC, reversal of the neutrophil/lymphocyte ratio and increase of total counts of eosinophils and basophils. From the age of six weeks of life, all of the studied haematological parameters were comparable to those of adult rabbits.

• MeSH
• aklimatizace MeSH
• bazofily fyziologie   MeSH
• eozinofily fyziologie   MeSH
• králíci krev   MeSH
• lymfocyty fyziologie   MeSH
• monocyty fyziologie   MeSH
• neutrofily fyziologie   MeSH
• počet erytrocytů * MeSH
• počet leukocytů * MeSH
• referenční hodnoty MeSH
• stárnutí fyziologie   MeSH
• těhotenství MeSH
• zvířata MeSH
• Check Tag
• králíci krev   MeSH
• mužské pohlaví MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The CD8(+) natural killer (NK) subpopulation has recently been identified as a fast and reliable biodosimetric indicator within human peripheral blood mononuclear cells (PBMC) in vitro. In irradiated and subsequently cultivated PBMC, a decrease of the relative number of intact CD3(-)CD8(+) lymphocytes 16 and 48 h after treatment has allowed for estimating the received dose in the range of 0 - 10 Gy and lethal/sublethal dose discrimination, respectively. Here we show that suitable biodosimeters can also be found in the peripheral blood B-cell compartment. Multiparameter flow cytometric analysis of irradiated and subsequently cultivated human PBMC revealed that both the CD27(+) and CD21(-) B-cell subpopulations can be used as biodosimeters and the CD19(+)CD27(+) lymphocytes have proved useful for retrospective determination of the received dose in the range of 0 - 6 Gy. In addition, several CD19(+) lymphocyte subsets characterized by co expression of CD21, CD27 and CD38 have been shown to bear biodosimetric potential, too. However, when important parameters like the original size within the CD19(+) compartment, its radiation-induced changes and data variation had been taken into account, the CD27(+) subpopulation proved superior to the other B-cell subpopulations and subsets. It appears that, in the dose range of 0 - 6 Gy, the relative decrease of CD27(+) B lymphocytes provides more sensitive and reliable data than that of CD8(+) NK-cells due mainly to lower data variation. In contrast to CD27(+) B cells, the proportions of CD27(+) subpopulations of T-cells were not affected by irradiation. We have also proposed a simple experimental protocol based on full blood cultivation and three-color CD27/CD3/CD19 immuno-phenotyping as a time-saving and inexpensive approach for practical biodosimetric evaluations on simple, three-to-four color flow cytometers.

• MeSH
• annexin A5 metabolismus   MeSH
• antigeny CD27 fyziologie   MeSH
• antigeny CD38 fyziologie   MeSH
• B-lymfocyty fyziologie   účinky záření   MeSH
• biologické markery MeSH
• fenotyp MeSH
• fosfatidylseriny metabolismus   MeSH
• lidé MeSH
• monocyty fyziologie   MeSH
• podskupiny lymfocytů fyziologie   MeSH
• průtoková cytometrie MeSH
• receptory buněčného povrchu metabolismus   MeSH
• receptory komplementu 3d metabolismus   MeSH
• separace buněk MeSH
• vztah dávky záření a odpovědi MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• annexin A5 MeSH
• antigeny CD27 MeSH
• antigeny CD38 MeSH
• biologické markery MeSH
• fosfatidylseriny MeSH
• receptory buněčného povrchu MeSH
• receptory komplementu 3d MeSH

v-myb oncogene of avian myeloblastosis virus (AMV) transforms myelomonocytic cells in vitro and induces acute monoblastic leukemia in vivo. The transforming effect of the v-myb can be suppressed using phorbol ester (TPA) or histone deacetylase inhibitor trichostatin A (TSA), the inducers of cell differentiation that are in clinical trials. In this study, we used proteomics-based approach to identify proteins with variable expression in differentiated BM2 cells. Proteome variations induced by TPA and TSA were compared to examine the mechanism of differentiation-promoting effects of these drugs. We found that expression of several proteins participating in cell cytoskeleton rearrangement, heat shock response, proteosynthesis and cell signaling was altered in TPA- or TSA-treated cells. We present here the first comparative proteome analysis of v-myb-transformed monoblasts BM2 focused on identification of proteins involved in their terminal differentiation.

• MeSH
• 2D gelová elektroforéza metody   MeSH
• buněčná diferenciace účinky léků   fyziologie   MeSH
• forbolové estery farmakologie   MeSH
• hmotnostní spektrometrie metody   MeSH
• kur domácí MeSH
• kyseliny hydroxamové farmakologie   MeSH
• monocyty účinky léků   fyziologie   MeSH
• onkogenní proteiny v-myb účinky léků   fyziologie   MeSH
• proteiny analýza   fyziologie   MeSH
• proteomika metody   MeSH
• transformované buněčné linie MeSH
• virus ptačí myeloblastózy fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• forbolové estery MeSH
• kyseliny hydroxamové MeSH
• onkogenní proteiny v-myb MeSH
• proteiny MeSH
• trichostatin A MeSH Prohlížeč

Maturation of blood cells depends on dramatic changes of expression profiles of specific genes. Although these changes have been extensively studied, their functional outcomes often remain unclear. In this study, we explored the identity and function of an unknown protein that was greatly overexpressed in v-myb-transformed BM2 monoblasts undergoing differentiation to macrophage-like cells. We identified this protein as vimentin, the intermediate filament protein. We show that an increased level of vimentin protein results from activation of the vimentin gene promoter occurring in monoblastic cells induced to differentiate by multiple agents. Furthermore, our studies reveal that the vimentin gene promoter is stimulated by Myb and Jun proteins, the key transcriptional regulators of myeloid maturation. Silencing of vimentin gene expression using siRNA markedly suppressed the ability of BM2 cells to form macrophage polykaryons active in phagocytosis and producing reactive oxygen species. Taken together, these findings document that up-regulation of vimentin gene expression is important for formation of fully active macrophage-like cells and macrophage polykaryons.

• MeSH
• 2D gelová elektroforéza MeSH
• buněčná diferenciace * MeSH
• fibroblasty MeSH
• geny jun genetika   MeSH
• hematopoéza genetika   MeSH
• hmotnostní spektrometrie MeSH
• křepelky a křepelovití MeSH
• kur domácí MeSH
• makrofágy cytologie   fyziologie   MeSH
• monocyty cytologie   fyziologie   MeSH
• onkogenní proteiny v-myb genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protoonkogenní proteiny c-jun fyziologie   MeSH
• regulace genové exprese MeSH
• tetradekanoylforbolacetát MeSH
• transformované buněčné linie MeSH
• transkripční faktory genetika   MeSH
• upregulace MeSH
• vimentin genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• onkogenní proteiny v-myb MeSH
• protoonkogenní proteiny c-jun MeSH
• tetradekanoylforbolacetát MeSH
• transkripční faktory MeSH
• vimentin MeSH

CREB-binding protein (CBP) regulates gene expression by binding to certain components of basal transcription machinery and by histone acetylation. In addition, it integrates various cellular signaling pathways through binding to multiple transcription factors, including the Myb proteins. We report in this study that CBP can partially suppress function of the v-Myb oncoprotein in leukemic cells. Although originally described as an activator of v-Myb function, we show that CBP can also act as a v-Myb suppressor. Ectopic expression of murine CBP in v-Myb-transformed chicken monoblasts reduced transcriptional activation abilities of the v-Myb protein and increased sensitivity to differentiation inducers such as phorbol ester or trichostatin A. In addition, exogenous CBP affected morphology of differentiated cells derived from BM2 monoblasts. These results indicate that cellular context is an important factor determining whether CBP will activate or suppress the protein it targets.

• MeSH
• buněčná diferenciace účinky léků   fyziologie   MeSH
• fagocytóza fyziologie   MeSH
• forbolové estery farmakologie   MeSH
• jaderné proteiny genetika   fyziologie   MeSH
• kur domácí MeSH
• kyseliny hydroxamové farmakologie   MeSH
• monocyty cytologie   účinky léků   fyziologie   MeSH
• myši MeSH
• onkogenní proteiny v-myb fyziologie   MeSH
• protein vázající CREB MeSH
• trans-aktivátory genetika   fyziologie   MeSH
• transformované buněčné linie MeSH
• virová transformace buněk fyziologie   MeSH
• virus ptačí myeloblastózy fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Crebbp protein, mouse MeSH Prohlížeč
• forbolové estery MeSH
• jaderné proteiny MeSH
• kyseliny hydroxamové MeSH
• onkogenní proteiny v-myb MeSH
• protein vázající CREB MeSH
• trans-aktivátory MeSH
• trichostatin A MeSH Prohlížeč

Cytokines play a major role in the control of inflammatory responses, participate in the regulation of blood phagocyte activities and as such are used for immunomodulatory therapy. In the present study, the influence of IL-10 on human blood phagocyte activity in the presence/absence of IL-6, IL-8 and TNF-alpha was tested in vitro. Our research analyzed the effects of cytokines on the production of reactive oxygen species measured by chemiluminescence and flow cytometry, and on the expression of surface molecules (CD11b, CD15, CD62L, CD31) measured by flow cytometry. IL-10 had no inhibitory effect on reactive oxygen species production and the expression of any examined adhesion molecule by resting or stimulated blood phagocytes within 3 h of incubation. Conversely, TNF-alpha, IL-6, and IL-8 increased reactive oxygen species production and the expression of CD11b and CD15 on both neutrophils and monocytes and decreased the expression of CD62L. These priming effects of the tested pro-inflammatory cytokines were not affected by IL-10. The obtained results suggest that IL-10 does not directly control blood phagocyte activation. These results also provide better information about the contribution of IL-6, IL-8 and TNF-alpha to the regulation of blood phagocyte-mediated inflammatory processes.

• MeSH
• antigen Lewis X metabolismus   MeSH
• antigeny CD11b metabolismus   MeSH
• antigeny CD31 metabolismus   MeSH
• časové faktory MeSH
• CD antigeny metabolismus   MeSH
• fagocyty účinky léků   metabolismus   fyziologie   MeSH
• interleukin-10 aplikace a dávkování   farmakologie   fyziologie   MeSH
• interleukin-6 aplikace a dávkování   farmakologie   MeSH
• interleukin-8 aplikace a dávkování   farmakologie   MeSH
• L-selektin metabolismus   MeSH
• leukocyty účinky léků   metabolismus   fyziologie   MeSH
• lidé MeSH
• luminiscenční měření MeSH
• monocyty účinky léků   metabolismus   fyziologie   MeSH
• neutrofily účinky léků   metabolismus   fyziologie   MeSH
• průtoková cytometrie MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• respirační vzplanutí účinky léků   fyziologie   MeSH
• rhodaminy chemie   MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• TNF-alfa aplikace a dávkování   farmakologie   MeSH
• zymosan farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigen Lewis X MeSH
• antigeny CD11b MeSH
• antigeny CD31 MeSH
• CD antigeny MeSH
• dihydrorhodamine 123 MeSH Prohlížeč
• interleukin-10 MeSH
• interleukin-6 MeSH
• interleukin-8 MeSH
• L-selektin MeSH
• reaktivní formy kyslíku MeSH
• rhodaminy MeSH
• tetradekanoylforbolacetát MeSH
• TNF-alfa MeSH
• zymosan MeSH

Wheat gliadin is the triggering agent in coeliac disease. In this study, we documented that proteolytic fragments of gliadin, in contrast to other food antigens, induced interleukin (IL)-8 and tumour necrosis factor-alpha (TNF-alpha) production and significantly increased interferon (IFN)-gamma-induced cytokine secretion in human monocytic line THP-1 cells. Stimulation with gliadin resulted in elevated phosphorylation of the IkappaBalpha molecule and increased NF-kappaB/DNA binding activity that was inhibited by sulfasalazine, l-1-tosylamido-2-phenylethyl chloromethyl ketone and pyrrolidine dithiocarbamate (PDTC). The activation pathway was shown to be independent of the CD14 molecule. Less mature U-937 monocytes responded to gliadin stimulation by low IL-8 secretion, TNF-alpha production was not detectable. We propose that gliadin-induced activation of monocytes/macrophages can participate in mechanisms leading to the impairment of intestinal mucosa in coeliac patients.

• MeSH
• buněčné linie MeSH
• ELISA MeSH
• gliadin farmakologie   MeSH
• interleukin-8 biosyntéza   MeSH
• kinetika MeSH
• lidé MeSH
• monocyty účinky léků   imunologie   fyziologie   MeSH
• NF-kappa B metabolismus   MeSH
• ovalbumin farmakologie   MeSH
• peptidové fragmenty farmakologie   MeSH
• proteiny ze sójových bobů farmakologie   MeSH
• pšenice MeSH
• sekvence aminokyselin MeSH
• TNF-alfa biosyntéza   MeSH
• U937 buňky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• gliadin MeSH
• interleukin-8 MeSH
• NF-kappa B MeSH
• ovalbumin MeSH
• peptidové fragmenty MeSH
• proteiny ze sójových bobů MeSH
• TNF-alfa MeSH