The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: Progress and Challenges in the Replacement of HIST' was held on 24 August 2014, in Prague, Czech Republic, as a satellite meeting to the 9th World Congress on Alternatives and Animal Use in the Life Sciences. Participants discussed the progress and challenges associated with the development, validation, and implementation of in vitro assays as replacements for the histamine sensitisation test (HIST) for acellular pertussis vaccines. Discussions focused on the consistency approach, the necessary framework for regulatory acceptance of a harmonised method, and recent international efforts towards the development of in vitro assays to replace the HIST. Workshop participants agreed that acceptable alternatives to the HIST should be based on ADP ribosylation-mediated cell intoxication and therefore that the CHO cell clustering assay, which measures cell intoxication, should be further pursued and developed as a possible replacement for the HIST. Participants also agreed to continue ongoing multinational discussions involving national and international standardisation authorities to reach consensus and to organise collaborative studies in this context for assay characterisation and calibration of reference materials.

• Klíčová slova
• Pertussis vaccines, histamine sensitisation test, replacement alternative,
• MeSH
• acelulární vakcíny terapeutické užití   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• histamin aplikace a dávkování   MeSH
• křečci praví MeSH
• lidé MeSH
• myši MeSH
• pertuse diagnóza   prevence a kontrola   MeSH
• pertusová vakcína terapeutické užití   MeSH
• pertusový toxin terapeutické užití   MeSH
• výchova a vzdělávání metody   trendy   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• kongresy MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• acelulární vakcíny MeSH
• histamin MeSH
• pertusová vakcína MeSH
• pertusový toxin MeSH

Human CMV infects between 50-85% of healthy individuals and can cause live-threatening infections in immunocompromised patients. Therefore, peptide vaccination is being developed as a promising immunotherapeutic approach for treatment of patients at risk of CMV disease. The enzymatically inactive toxoid of Bordetella adenylate cyclase (CyaA-AC(-)) was shown to be an efficient tool for delivery of peptide epitopes and stimulation of Ag-specific T-cell immune responses. We investigated here the capacity of two CyaA-AC(-) constructs to deliver epitopes derived from the CMV phosphoprotein pp65 for activation of human T cells in vitro. Expansion of γ-IFN-secreting CMV-specific CD8(+) T cells, as well as increase of total IFN-γ and TNF-α production by PBMCs from CMV-seropositive donors were observed after in vitro stimulation with CyaA-AC(-) constructs carrying CMV epitopes, whereas limited activation of immune response occurred with free peptides. The activation of immune response was confirmed by expansion of CMV-specific T-cell clones and anti-CMV cytotoxic effect of stimulated PBMCs. These data open the way to clinical evaluation of CyaA-AC(-) constructs as tools for detection and expansion of CMV-specific T-cell immune responses for diagnostic and immunotherapeutic applications against CMV-associated diseases.

• MeSH
• adenylátcyklasy genetika   imunologie   MeSH
• aktivace lymfocytů MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• Cytomegalovirus imunologie   MeSH
• epitopy T-lymfocytární imunologie   MeSH
• fosfoproteiny imunologie   MeSH
• lidé MeSH
• peptidové fragmenty genetika   imunologie   MeSH
• proteiny virové matrix imunologie   MeSH
• sekvence aminokyselin MeSH
• vakcíny proti cytomegalovirové infekci genetika   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH
• cytomegalovirus matrix protein 65kDa MeSH Prohlížeč
• epitopy T-lymfocytární MeSH
• fosfoproteiny MeSH
• peptidové fragmenty MeSH
• proteiny virové matrix MeSH
• vakcíny proti cytomegalovirové infekci MeSH

The Neisseria meningitidis FAM20 strain secretes two proteins of unknown function, FrpA and FrpC, which contain typical RTX domains found in cytotoxins from other gram-negative pathogens. To evaluate whether the Frp proteins could be involved in meningococcal virulence, 65 isolates of all serogroups were screened by PCR for the presence of both frp genes. The frpA allele was, however, poorly conserved. A single strain harbored an frpA allele of the previously described size, while large insertions were detected in the frpA loci of 22 isolates (34%), and the 42 remaining isolates (65%) did not contain frpA at all. In contrast, frpC alleles, albeit of variable length, were detected in all invasive and most carrier strains. This suggests that meningococci may produce a family of FrpC proteins of various molecular masses. High levels of both immunoglobulin G (IgG) and IgA class antibodies recognizing recombinant FrpC were indeed detected in convalescent-phase sera of most patients at 2 and 4 to 5 weeks after the first symptoms of meningococcal disease. These results show that FrpC-like proteins are produced and may play a role in invasive meningococcal infections.

• MeSH
• alely MeSH
• bakteriální proteiny genetika   imunologie   izolace a purifikace   metabolismus   MeSH
• cytotoxiny * MeSH
• klonování DNA MeSH
• lidé MeSH
• membránové proteiny * MeSH
• meningokokové infekce imunologie   mikrobiologie   MeSH
• molekulární sekvence - údaje MeSH
• Neisseria meningitidis imunologie   metabolismus   MeSH
• periplazmatické vazebné proteiny MeSH
• polymorfismus genetický genetika   MeSH
• proteiny vázající železo MeSH
• proteiny vnější bakteriální membrány MeSH
• protilátky bakteriální krev   MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• cytotoxiny * MeSH
• frpC protein, Neisseria meningitidis MeSH Prohlížeč
• membránové proteiny * MeSH
• periplazmatické vazebné proteiny MeSH
• proteiny vázající železo MeSH
• proteiny vnější bakteriální membrány MeSH
• protilátky bakteriální MeSH
• rekombinantní proteiny MeSH

The capacity of adenylate cyclase toxin (ACT) to penetrate into target cells depends on post-translational fatty-acylation by the acyltransferase CyaC, which can palmitoylate the conserved lysines 983 and 860 of ACT. Here, the in vivo acylating capacity of a set of mutated CyaC acyltransferases was characterized by two-dimensional gel electrophoresis and mass spectrometric analyses of the ACT product. Substitutions of the potentially catalytic serine 20 and histidine 33 residues ablated acylating activity of CyaC. Conservative replacements of alanine 140 by glycine (A140G) and valine (A140V) residues, however, affected selectivity of CyaC for the two acylation sites on ACT. Activation by the A140G variant of CyaC generated a mixture of bi- and monoacylated ACT molecules, modified either at both Lys-860 and Lys-983, or only at Lys-860, respectively. In contrast, the A140V CyaC produced a nearly 1:1 mixture of nonacylated pro-ACT with ACT monoacylated almost exclusively at Lys-983. The respective proportion of toxin molecules acylated at Lys-983 correlated well with the cell-invasive activity of both ACT mixtures, which was about half of that of ACT fully acylated on Lys-983 by intact CyaC. These results show that acylation of Lys-860 alone does not confer cell-invasive activity on ACT, whereas acylation of Lys-983 is necessary and sufficient.

• MeSH
• 2D gelová elektroforéza MeSH
• acylace MeSH
• acyltransferasy chemie   genetika   metabolismus   MeSH
• adenylátcyklasový toxin * MeSH
• Bordetella pertussis enzymologie   MeSH
• erytrocyty účinky léků   metabolismus   patologie   MeSH
• faktory virulence rodu Bordetella chemie   genetika   metabolismus   toxicita   MeSH
• hemolýza účinky léků   MeSH
• histidin genetika   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• kyselina palmitová metabolismus   MeSH
• lysin genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• ovce MeSH
• peptidové fragmenty chemie   metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• serin genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• acyltransferasy MeSH
• adenylátcyklasový toxin * MeSH
• faktory virulence rodu Bordetella MeSH
• histidin MeSH
• kyselina palmitová MeSH
• lysin MeSH
• peptidové fragmenty MeSH
• serin MeSH

Calreticulin (CRT), a high-affintiy calcium binding protein and chaperone, was recently identified as one of the targets of autoantibodies in coeliac disease. We evaluated the level of IgA and IgG antibodies to CRT in sera from patients with coeliac disease and various autoimmune diseases. The level of antibodies to gliadin (shown previously to cross-react with CTR), isolated enterocytes and tissue transglutaminase were determined for comparison. The mean level of IgA antibodies to CRT was significantly higher (P< 0.001) in sera from coeliac patients with active disease (139.9+/-11.2 AU/+/-SE) than in healthy controls (20.9+/-1.7 AU). In sera of patients with systemic lupus erythematosus (SLE), insulin dependent diabetes mellitus (IDDM), multiple sclerosis (MS) and autoimmune thyroiditis (AT) or inflammatory bowel disease (IBD) the mean level (25.8+/-3.7 to 38.1+/-5.6 AU) did not exceed the cut-off value. A low level of these antibodies, however, was detected in some sera of patients with MS and IBD. The level of IgG anti-CRT antibodies was increased in coeliac patients (mean 125.4+/-8.0 AU, P< 0.001) when compared to that in healthy controls (33.9+/-2.3 AU). The IgG anti-CRT antibodies were also detected in about 30% of SLE patients sera (54.1+/-3.6 AU, P< 0.001), but the mean level reached only half that detected in coeliac patients.

• MeSH
• autoimunitní nemoci imunologie   MeSH
• autoprotilátky krev   MeSH
• celiakie imunologie   MeSH
• dítě MeSH
• dospělí MeSH
• ELISA MeSH
• imunoglobulin A krev   MeSH
• imunoglobulin G krev   MeSH
• kalretikulin MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• předškolní dítě MeSH
• proteiny vázající vápník imunologie   MeSH
• ribonukleoproteiny imunologie   MeSH
• senioři MeSH
• western blotting MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• předškolní dítě MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoprotilátky MeSH
• imunoglobulin A MeSH
• imunoglobulin G MeSH
• kalretikulin MeSH
• proteiny vázající vápník MeSH
• ribonukleoproteiny MeSH

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (ACT-Hly) can penetrate a variety of eukaryotic cells. Recombinant AC toxoids have therefore been recently used for delivery of CD8(+) T-cell epitopes into antigen-presenting cells in vivo and for induction of protective antiviral, as well as therapeutic antitumor cytotoxic T-cell responses. We have explored the carrier potential of the ACT molecule by insertional mutagenesis scanning for new permissive sites, at which integration of two- to nine-residue-long peptides does not interfere with membrane interaction and translocation of ACT. A model CD8(+) T-cell epitope of ovalbumin was incorporated at 10 of these permissive sites along the toxin molecule, and the capacity of ACT constructs to penetrate into cell cytosol and deliver the epitope into the major histocompatibility complex (MHC) class I antigen processing and presentation pathway was examined. While all six constructs bearing the epitope within the Hly portion of ACT failed to deliver the epitope to the MHC class I molecules, all four toxoids with inserts within different permissive sites in the AC domain efficiently delivered the epitope into this cytosolic pathway, giving rise to stimulation of a specific CD8(+) T-cell hybridoma. The results suggest that, in contrast to the AC domain, the hemolysin moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.

• MeSH
• adenylátcyklasový toxin MeSH
• adenylátcyklasy genetika   imunologie   metabolismus   MeSH
• bakteriální proteiny genetika   imunologie   metabolismus   MeSH
• Bordetella pertussis enzymologie   genetika   imunologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• DNA primery genetika   MeSH
• epitopy aplikace a dávkování   MeSH
• faktory virulence rodu Bordetella genetika   imunologie   metabolismus   MeSH
• hemolyziny genetika   imunologie   metabolismus   MeSH
• inzerční mutageneze MeSH
• MHC antigeny I. třídy metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• prezentace antigenu * MeSH
• proteinové prekurzory genetika   imunologie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• techniky in vitro MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• adenylátcyklasy MeSH
• bakteriální proteiny MeSH
• DNA primery MeSH
• epitopy MeSH
• faktory virulence rodu Bordetella MeSH
• hemolyziny MeSH
• MHC antigeny I. třídy MeSH
• proteinové prekurzory MeSH

The Bordetella pertussis adenylate cyclase toxin-hemolysin (ACT or CyaA) is a multifunctional protein. It forms small cation-selective channels in target cell and lipid bilayer membranes and it delivers into cell cytosol the amino-terminal adenylate cyclase (AC) domain, which catalyzes uncontrolled conversion of ATP to cAMP and causes cell intoxication. Here, we demonstrate that membrane translocation of the AC domain into cells is selectively dissociated from ACT membrane insertion and channel formation when a helix-breaking proline residue is substituted for glutamate 509 (Glu-509) within a predicted transmembrane amphipathic alpha-helix. Neutral substitutions of Glu-509 had little effect on toxin activities. In contrast, charge reversal by lysine substitutions of the Glu-509 or of the adjacent Glu-516 residue reduced the capacity of the toxin to translocate the AC domain across membrane and enhanced significantly its specific hemolytic activity and channel forming capacity in lipid bilayer membranes. Combination of the E509K and E516K mutations in a single molecule further exacerbated hemolytic and channel forming activity and ablated translocation of the AC domain into cells. The lysine substitutions strongly decreased the cation selectivity of the channels, indicating that Glu-509 and Glu-516 are located within or close to the membrane channel. These results suggest that the structure including glutamate residues 509 and 516 is critical for AC membrane translocation and channel forming activity of ACT.

• MeSH
• adenylátcyklasový toxin * MeSH
• biologický transport MeSH
• bodová mutace MeSH
• buněčná membrána metabolismus   MeSH
• DNA primery MeSH
• faktory virulence rodu Bordetella genetika   metabolismus   farmakologie   MeSH
• hemolýza účinky léků   MeSH
• iontové kanály metabolismus   MeSH
• kationty MeSH
• kyselina glutamová genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• ovce MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• DNA primery MeSH
• faktory virulence rodu Bordetella MeSH
• iontové kanály MeSH
• kationty MeSH
• kyselina glutamová MeSH

Bordetella pertussis adenylate cyclase toxin (ACT) is one of the few known protein toxins penetrating directly into the cytosol of target cells across their cytoplasmic membrane without the need for endocytosis. This capacity of ACT was recently exploited for in vivo delivery of single viral CD8(+) T-epitopes into MHC class I-presenting cells and induction of protective antiviral cytotoxic T-cell (CTL) responses. Here, we have explored the potential of the cell-invasive adenylate cyclase domain of the toxin to deliver larger antigens by evaluating the epitope-specific CTL responses induced by constructs bearing one to four copies of the CD8(+) T-epitope from the nucleoprotein of the lymphocytic choriomeningitis virus. The increase in the number of copies of the epitope was accompanied by a moderate decrease of the specific cell invasiveness of the ACT protein and did not lead to further enhancement of the level of induced epitope-specific CTL cells in mice, as compared to ACT with a single copy of the epitope. These results demonstrate the capacity of ACT to deliver larger heterologous antigens comprising several epitopes for antigenic presentation in vivo.

• MeSH
• adenylátcyklasový toxin * MeSH
• antigeny CD8 genetika   MeSH
• Bordetella pertussis chemie   MeSH
• cytotoxicita imunologická MeSH
• epitopy MeSH
• Escherichia coli metabolismus   MeSH
• faktory virulence rodu Bordetella biosyntéza   genetika   imunologie   MeSH
• myši inbrední BALB C imunologie   MeSH
• myši MeSH
• rekombinantní proteiny biosyntéza   genetika   imunologie   MeSH
• T-lymfocyty imunologie   MeSH
• vakcinace MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• antigeny CD8 MeSH
• epitopy MeSH
• faktory virulence rodu Bordetella MeSH
• rekombinantní proteiny MeSH

The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the epsilon-amino group of lysine 983 (Lys983) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys860), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additional acylation site by replacing Lys860 of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of Lys860 did not affect the quantitative acylation of Lys983 by palmitoyl (C16:0) and palmitoleil (cis Delta9 C16:1) fatty-acyl groups. Nevertheless, even the most conservative substitution of lysine 860 by an arginine residue caused a 10-fold decrease of toxin activity. This resulted from a 5-fold reduction of cell association capacity and a further 2-fold reduction in cell penetration efficiency of the membrane-bound K860R toxin. These results suggest that lysine 860 plays by itself a crucial structural role in membrane insertion and translocation of the toxin, independently of its acylation status.

• MeSH
• acylace MeSH
• adenylátcyklasový toxin MeSH
• adenylátcyklasy chemie   metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• Bordetella pertussis enzymologie   MeSH
• DNA primery MeSH
• faktory virulence rodu Bordetella chemie   genetika   metabolismus   MeSH
• konzervovaná sekvence MeSH
• lysin chemie   metabolismus   MeSH
• mastné kyseliny metabolismus   MeSH
• mutageneze cílená MeSH
• proteinové prekurzory chemie   genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• adenylátcyklasy MeSH
• bakteriální proteiny MeSH
• DNA primery MeSH
• faktory virulence rodu Bordetella MeSH
• lysin MeSH
• mastné kyseliny MeSH
• proteinové prekurzory MeSH

• MeSH
• bakteriální infekce mikrobiologie   MeSH
• biologie trendy   MeSH
• eukaryotické buňky cytologie   mikrobiologie   MeSH
• lidé MeSH
• prokaryotické buňky cytologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH