The protective effect of Enterococcus faecium EFAL41 on chicken's caecum in relation to the TLR (TLR4 and TLR21) activation and production of luminal IgA challenged with Campylobacter jejuni CCM6191 was assessed. The activation of MIF, IFN-β, MD-2 and CD14 was followed-up after bacterial infection. Day-old chicks (40) were divided into four groups (n = 10): control (C), E. faecium AL41 (EFAL41), C. jejuni (CJ) and combined E. faecium AL41+C. jejuni (EFAL41+CJ). Relative mRNA expression of TLR4, TLR21 and CD14 was upregulated in the probiotic strain and infected (combined) group on day 4 and 7 post infection (p.i.). The caecal relative MD-2 mRNA expression was upregulated on day 4 p.i. in the EFAL41+CJ and CJ groups. MIF and IFN-β reached the highest levels in the combined groups on day 7 p.i. The concentration of the sIgA in intestinal flush was upregulated in EFAL41+CJ group on day 4 p.i. The results demonstrated that E. faecium EFAL41 probiotic strain can modulate the TLRs expression and modify the activation of MIF, IFN-β, MD-2 and CD14 molecules in the chickens caecum challenged with C. jejuni CCM 6191. The counts of EFAL41 were sufficient and high, similarly the counts of enterococci in both, caecum and faeces but without reduction of Campylobacter counts.
- MeSH
- antigeny CD14 genetika imunologie MeSH
- Campylobacter jejuni růst a vývoj MeSH
- cékum imunologie mikrobiologie MeSH
- Enterococcus faecium růst a vývoj imunologie MeSH
- feces mikrobiologie MeSH
- imunoglobulin A sekreční genetika MeSH
- interakce hostitele a patogenu MeSH
- interferon beta genetika imunologie MeSH
- kampylobakterové infekce dietoterapie imunologie mikrobiologie veterinární MeSH
- kur domácí MeSH
- lymfocytární antigen 96 genetika imunologie MeSH
- nemoci drůbeže dietoterapie genetika imunologie mikrobiologie MeSH
- novorozená zvířata MeSH
- probiotika farmakologie MeSH
- protein - isoformy genetika imunologie MeSH
- receptory imunologické genetika imunologie MeSH
- regulace genové exprese MeSH
- signální transdukce MeSH
- toll-like receptor 4 genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The interleukin 17 (IL-17) cytokine and receptor family is central to antimicrobial resistance and inflammation in the lung. Mice lacking IL-17A, IL-17F, or the IL-17RA subunit were compared with wild-type mice for susceptibility to airway inflammation in models of infection and allergy. Signaling through IL-17RA was required for efficient microbial clearance and prevention of allergy; in the absence of IL-17RA, signaling through IL-17RC on epithelial cells, predominantly by IL-17F, significantly exacerbated lower airway Aspergillus or Pseudomonas infection and allergic airway inflammation. In contrast, following infection with the upper respiratory pathogen Staphylococcus aureus, the IL-17F/IL-17RC axis mediated protection. Thus, IL-17A and IL-17F exert distinct biological effects during pulmonary infection; the IL-17F/IL-17RC signaling axis has the potential to significantly worsen pathogen-associated inflammation of the lower respiratory tract in particular, and should be investigated further as a therapeutic target for treating pathological inflammation in the lung.
- MeSH
- alergie genetika imunologie mikrobiologie patologie MeSH
- Aspergillus imunologie MeSH
- aspergilóza genetika imunologie mikrobiologie patologie MeSH
- epitelové buňky imunologie mikrobiologie patologie MeSH
- interleukin-17 nedostatek genetika imunologie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- náchylnost k nemoci MeSH
- plíce imunologie mikrobiologie patologie MeSH
- protein - isoformy nedostatek genetika imunologie MeSH
- pseudomonádové infekce genetika imunologie mikrobiologie patologie MeSH
- Pseudomonas imunologie MeSH
- receptory interleukinu-17 nedostatek genetika imunologie MeSH
- regulace genové exprese MeSH
- respirační sliznice imunologie mikrobiologie patologie MeSH
- signální transdukce MeSH
- stafylokokové infekce genetika imunologie mikrobiologie patologie MeSH
- Staphylococcus aureus imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The aim of the present study is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ~4 µm in diameter and containing ∼1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS/PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.
- MeSH
- imobilizované proteiny chemie imunologie MeSH
- imunoglobulin G imunologie izolace a purifikace MeSH
- lidé MeSH
- magnetismus metody MeSH
- magnety chemie MeSH
- mikrosféry MeSH
- myosin typu I chemie imunologie MeSH
- myši MeSH
- polyhydroxyethylmethakrylát chemie MeSH
- protein - isoformy chemie imunologie MeSH
- roztroušená skleróza diagnóza imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1β (IL-1β) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.
- MeSH
- Anaplasma phagocytophilum genetika imunologie MeSH
- annexin A2 chemie genetika imunologie MeSH
- arachnida jako vektory chemie genetika imunologie MeSH
- cystatiny chemie genetika imunologie MeSH
- ehrlichióza imunologie mikrobiologie patologie MeSH
- Escherichia coli genetika metabolismus MeSH
- imunitní únik * MeSH
- inflamasomy genetika imunologie MeSH
- interleukin-18 genetika imunologie MeSH
- interleukin-1beta genetika imunologie MeSH
- kaspasa 1 genetika imunologie MeSH
- kaspasy genetika imunologie MeSH
- klíště chemie genetika imunologie MeSH
- lidé MeSH
- makrofágy imunologie mikrobiologie MeSH
- molekulární modely MeSH
- myši MeSH
- protein - isoformy chemie genetika imunologie MeSH
- proteiny regulující apoptózu chemie genetika imunologie MeSH
- proteiny vázající vápník chemie genetika imunologie MeSH
- regulace genové exprese MeSH
- rekombinantní proteiny chemie genetika imunologie MeSH
- sekvence aminokyselin MeSH
- signální transdukce MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Polymorphisms of genes involved in innate and adaptive immunity have become an object of major interest in regard to hematopoietic stem cell transplantation (HSCT) complications. Regimen-related gastrointestinal toxicity (RR-GIT) is the dominant complication during the pre-engraftment period and has been linked to increased risk of graft-versus-host disease (GVHD) development. According to our hypothesis, functional variants of genes participating in DNA damage response (DDR) may have an impact on the extent of tissue damage caused by the conditioning regimen. In our single-center study, we analyzed 62 patients who underwent HSCT from HLA-identical donors after reduced conditioning. The patients were genotyped for 5 single nucleotide polymorphisms (SNPs, rs4585 T/G, rs189037 A/G, rs227092 T/G, rs228590 C/T, and rs664677 T/C) of the ATM gene-the essential member of the DDR pathways, using allele-specific matrix-assisted laser desorption/ionization, time-of-flight (MALDI-TOF) mass spectrometry assay. Because of almost absolute linkage disequilibrium observed among all 5 SNPs, association of 2 major ATM haplotypes (ATM1/ATM2) with RR-GIT and acute GVHD (aGVHD) was analyzed. Importantly, the univariate and multivariate analysis showed that patients homozygous for ATM2 haplotype (rs4585*T, rs189037*A, rs227092*T, rs228590*C, and rs664677*T) are more likely to suffer from high-grade RR-GIT than ATM1 homozygous patients. The association with aGVHD was not significant. To our knowledge, this is the first report showing the ATM gene variability in relation to RR-GIT in the allogeneic HSCT setting.
- MeSH
- akutní nemoc MeSH
- alely MeSH
- analýza přežití MeSH
- ATM protein genetika imunologie MeSH
- dárci tkání MeSH
- dospělí MeSH
- exprese genu MeSH
- frekvence genu MeSH
- gastrointestinální trakt účinky léků imunologie patologie MeSH
- haplotypy MeSH
- hematologické nádory genetika imunologie mortalita terapie MeSH
- homologní transplantace MeSH
- jednonukleotidový polymorfismus * MeSH
- lidé středního věku MeSH
- lidé MeSH
- myeloablativní agonisté aplikace a dávkování škodlivé účinky MeSH
- nemoc štěpu proti hostiteli genetika imunologie mortalita MeSH
- příprava pacienta k transplantaci metody MeSH
- prognóza MeSH
- prospektivní studie MeSH
- protein - isoformy genetika imunologie MeSH
- transplantace hematopoetických kmenových buněk * MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Recognition of glycosylation patterns is one of the basic features of innate immunity. Ability of C-type lectin-like receptors such as NKR-P1 to bind saccharide moieties has become recently a controversial issue. In the present study, binding assay with soluble fluorescently labeled recombinant rat NKR-P1A and mouse NKR-P1C proteins revealed apparently no affinity to the various neoglycoproteins. Lack of functional linkage between NKR-P1 and previously described saccharide binder was supported by the fact, that synthetic N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P) did not change gene expression of NKR-P1 isoforms in C57BL/6 and BALB/c mice divergent in the NK gene complex (both in vitro and in vivo). Surprisingly, N-acetyl-D-glucosamine-coated tetrabranched polyamido-amine dendrimer specifically binds to NKT cells and macrophages but not to NK cells (consistently with changes in cytokine patterns). Despite the fact that GN8P has been tested as an immunomodulator in anti-cancer treatment animal models for many years, surprisingly no changes in cytokine profiles in serum relevant to anti-cancer responses using B16F10 and CT26 harboring mouse strains C57BL/6 and BALB/c are observed. Our results indicate possible indirect involvement of NK cells in GN8P mediated immune responses.
- MeSH
- acetylglukosamin imunologie metabolismus MeSH
- buňky NK imunologie metabolismus MeSH
- dendrimery metabolismus MeSH
- experimentální nádory farmakoterapie genetika imunologie MeSH
- exprese genu účinky léků imunologie MeSH
- glykokonjugáty imunologie metabolismus farmakologie MeSH
- interferon gama krev genetika imunologie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lektinové receptory NK-buněk - podrodina B genetika imunologie metabolismus MeSH
- lektiny typu C genetika imunologie metabolismus MeSH
- makrofágy imunologie metabolismus MeSH
- myši inbrední BALB C MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- NKT buňky imunologie metabolismus MeSH
- oligosacharidy imunologie metabolismus MeSH
- polyaminy imunologie metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protein - isoformy genetika imunologie metabolismus MeSH
- průtoková cytometrie MeSH
- slezina cytologie imunologie metabolismus MeSH
- TNF-alfa krev genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Microtubules formed by αβ-tubulin dimers represent cellular structures that are indispensable for the maintenance of cell morphology and for cell motility generation. Microtubules in intact cells are in highly regulated equilibrium with cellular pools of soluble tubulin dimers. Sensitive, reproducible and rapid assays are necessary to monitor tubulin changes in cytosolic pools after treatment with anti-mitotic drugs, during the cell cycle or activation and differentiation events. Here we describe new assays for α-tubulin quantification. The assays are based on sandwich ELISA, and the signal is amplified with biotinyl-tyramide or immuno-PCR. Matching monoclonal antibody pair recognizes phylogenetically highly conserved epitopes localized outside the C-terminal isotype-defining region. This makes it possible to detect α-tubulin isotypes in different cell types of various species. Biotinyl-tyramide amplification and immuno-PCR amplification enable detection of tubulin at concentrations 2.5ng/ml and 0.086ng/ml, respectively. Immuno-PCR detection shows enhanced sensitivity and wider dynamic range when compared to ELISA with biotinyl-tyramide detection. Our results on taxol-treated and activated bone marrow-derived mast cells demonstrate, that the assays allow sensitive quantification of tubulin in complex biological fluids.
- MeSH
- biotin analogy a deriváty MeSH
- buněčné linie MeSH
- ELISA metody statistika a číselné údaje MeSH
- mapování epitopu MeSH
- mastocyty účinky léků metabolismus MeSH
- monoklonální protilátky MeSH
- myši MeSH
- paclitaxel farmakologie MeSH
- polymerázová řetězová reakce metody MeSH
- protein - isoformy analýza genetika imunologie MeSH
- thapsigargin farmakologie MeSH
- tubulin analýza genetika imunologie MeSH
- tyramin analogy a deriváty MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The immune system of ticks is stimulated to produce many pharmacologically active molecules during feeding and especially during pathogen invasion. The family of cationic peptides - defensins - represents a specific group of antimicrobial compounds with six conserved cysteine residues in a molecule. RESULTS: Two isoforms of the defensin gene (def1 and def2) were identified in the European tick Ixodes ricinus. Expression of both genes was induced in different tick organs by a blood feeding or pathogen injection. We have tested the ability of synthetic peptides def1 and def2 to inhibit the growth or directly kill several pathogens. The antimicrobial activities (expressed as minimal inhibition concentration and minimal bactericidal concentration values) against Gram positive bacteria were confirmed, while Gram negative bacteria, yeast, Tick Borne Encephalitis and West Nile Viruses were shown to be insensitive. In addition to antimicrobial activities, the hemolysis effect of def1 and def2 on human erythrocytes was also established. CONCLUSIONS: Although there is nothing known about the realistic concentration of defensins in I. ricinus tick body, these results suggest that defensins play an important role in defence against different pathogens. Moreover this is a first report of a one amino acid substitution in a defensins molecule and its impact on antimicrobial activity.
- MeSH
- anatomické struktury zvířat imunologie MeSH
- antiinfekční látky izolace a purifikace farmakologie MeSH
- defensiny genetika imunologie izolace a purifikace MeSH
- erytrocyty účinky léků MeSH
- gramnegativní bakterie účinky léků MeSH
- grampozitivní bakterie účinky léků MeSH
- klíště genetika imunologie MeSH
- kvasinky účinky léků MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- morčata MeSH
- protein - isoformy genetika imunologie izolace a purifikace MeSH
- stanovení celkové genové exprese MeSH
- viry účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- morčata MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18) (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20) detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr(18) was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser(20) and Thr(18) kinase signalling cascades to modulate p53 activity.
- MeSH
- buněčné linie MeSH
- epitopy imunologie metabolismus MeSH
- fosfatasy metabolismus MeSH
- fosforylace MeSH
- fosfoserin imunologie metabolismus MeSH
- fosfothreonin imunologie metabolismus MeSH
- izoelektrický bod MeSH
- jaderné proteiny * MeSH
- lidé MeSH
- monoklonální protilátky imunologie MeSH
- nádorový supresorový protein p53 genetika imunologie metabolismus MeSH
- nádory prsu imunologie metabolismus MeSH
- peptidové fragmenty chemická syntéza imunologie metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- protein - isoformy genetika imunologie metabolismus MeSH
- protoonkogenní proteiny c-mdm2 MeSH
- protoonkogenní proteiny * metabolismus MeSH
- rekombinantní proteiny imunologie metabolismus MeSH
- sekvence aminokyselin MeSH
- signální transdukce MeSH
- specificita protilátek MeSH
- Spodoptera MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH