Cieľ práce: Od roku 1980 sa stali infekcie kmeňmi Escherichia coli produkujúcimi shiga toxíny (STEČ) veľmi sledovanými nielen mikrobiológmi ale aj širokou verejnosťou. STEČ sú asociované so závažným a často aj fatálne prebiehajúcim hemolyticko-uremickým syndrómom. Najdôležitejšiu úlohu v patogeneze tohoto ochorenia zohráva uvoľnenie shiga toxínov (Stx) do cirkulácie po prieniku cez črevnú stenu. STEČ získa najčastejšie pacient požitím kontaminovanej potravy. Autori opisujú rodinný výskyt hemolyticko-uremického syndrómu (HUS) spôsobeného kmeňom E. coli O157. Zo štrnástich členov rodiny, bola u deviatich členov detekovaná E. coli O157 v stolici, v troch prípadoch došlo k rozvoju HUS. Materiál a metódy: Bolo vyšetřených 69 vzoriek: 54 stolíc, 1 vzorka kozího mUeka a kozího trusu, 1 vzorka domácky vyrobenej detskej výživy, 7 vzoriek nepasterizovaného kravského mheka a 5 vzoriek kravského trusu. Na izoláciu STEČ a detekciu vybraných faktorov virulencie bola použitá selektívna kultivácia, imunomagnetická separácia, latexová aglutinácia a multiplexná PCR. Výsledky: Izolovali sme 14 kmeňov E. coli O157, ktoré nefermentovali sorbitol. Izolovali sme ich od pacientov s HUS (n = 3), s hemoragickou kolitidou (HC) (n = 2), od asymptomatických nosičov (n = 4), z kravského trusu (n = 4) a z kravského mlieka (n = 1). Všetky izolované kmene mali gén pre shiga toxin 2, intimín a enterohemolyzín. U vrtkých kmeňov sme dokázali produkciu Stx2. Záver. Autori detekovali prvýkrát na Slovensku STEČ produkujúce Stx2, ktoré vyvolali HUS. Autorom sa podarilo dokázať aj zdroj infekcie, kterým bolo kontaminované nepasterizované mlieko použité pri príprave pudingu.

Purpose of the study. Since 1980 both microbiologists and the general public are paying increasingly more attention to infections by the serotypes of Escherichia coli that produce shiga toxins (STEC). STECs are associated with the serious and often fatal haemolytic uraemic syndrome. Crucial in the pathogenesis of this disease is the release, through the intestinal wall, of shiga toxins (Stx) into blood circulation. The most frequent source of STECs is contaminated food. The authors describe the family incidence of the haemolytic uraemic syndrome (HUS) caused by the E. coli serotype O157. E. coli O157 was detected in the faeces of nine out of fourteen family members. HUS developed in three cases. Material and methods: 69 samples were analysed: 54 faeces samples, 1 sample of goat's milk and of goat's dung, 1 sample of home-made baby food, 7 samples of non-pasteurized cow's milk and 7 samples of cow's dung. Selective cultivation, immunomagnetic separation, latex agglutination and multiplex PCR were used to isolate STECs and to detect selected virulence factors. Results: We isolated 14 E. coli serotypes O157 that did not ferment sorbitol. They were isolated from patients presenting HUS (n = 3) and haemorrhagic colitis (HC) (n = 2), from asymptomatic carriers (n = 4), from cow's dung (n = 4) and from cow's milk (n = 1). All the isolated serotypes presented a gene for shiga toxin 2, intimin and enterohaemolysin. In all serotypes we demonstrated the production ofStx2. Conclusions: The authors detected, for the first time in Slovakia, STECs producing Stx2 that cause HUS. They succeeded in tracing the source of the infection -contaminated non-pasteurized milk used to make a pudding.

• MeSH
• bakteriologické techniky metody   MeSH
• Escherichia coli O157 patogenita   MeSH
• finanční podpora výzkumu jako téma MeSH
• hemolyticko-uremický syndrom diagnóza   patologie   MeSH
• imunologické techniky metody   MeSH
• infekce vyvolané Escherichia coli diagnóza   patologie   MeSH
• lidé MeSH
• shiga toxin 2 MeSH
• zdraví rodiny MeSH
• Check Tag
• lidé MeSH

Jako botulotoxin označujeme skupinu velmi podobných, vysoce toxických proteinů izolovaných z anaerobní bakterie Clostridium botulinum, která se nachází v půdě a může růst v mnoha potravinách. Botulotoxin účinkuje rychle, symptomy otravy se objevují obvykle mezi 18. až 36. hodinou po ingesci závadné potraviny. Ke smrti dochází prvý až třetí den. Botulotoxin blokuje neuromuskulámí přenos nervového vzruchu vazbou na receptorová místa nervosvalové ploténky, kde inhibuje uvolňování acetylcholinu. U vysokých dávek botulotoxinu to vede k respirační paralýze, která může trvat několik měsíců. Botulotoxin je vehni nebezpečná látka, která může být zneužita k vojenským či teroristickým útokům. Botulotoxin má však své uplamění v medicíně. Může být použit jako léčivo mnoha chorob způsobených svalovou hyperaktivitou.

Botulinum toxin is a group of related, highly poisonous protein agents isolated from fermentation of the anaerobic bacterium Clostridium botulinum, which naturally occurs in soil and can grow in many meats and vegetables. Botulinum toxin is fast-acting, usually producing symptoms within 18 to 36 hours after ingestion. Death occurs after one to three days. Botulinum toxin blocks neuromuscular conduction by bindmg to receptor sites on motor nerve terminals and by inhibiting the release of acetylcholine. Symptoms at high exposure levels can include respiratory distress and respiratory paralysis, which may persist several months. Botulinum toxin is very dangerous compound which can be misused to military or terrorist attack. In fact, botulinum toxin has conventional medical therapeutic uses. It can be used as therapeutic in many diseases due to muscle hyperactivity.

• MeSH
• botulotoxiny farmakologie   fyziologie   terapeutické užití   MeSH
• Clostridium botulinum patogenita   MeSH
• hodnocení rizik MeSH
• nervosvalové látky farmakologie   MeSH
• neuromuskulární nemoci patologie   terapie   MeSH

The mucus layer protects airway epithelia from damage by noxious agents. Intriguingly, Bordetella pertussis bacteria provoke massive mucus production by nasopharyngeal epithelia during the initial coryza-like catarrhal stage of human pertussis and the pathogen transmits in mucus-containing aerosol droplets expelled by sneezing and post-nasal drip-triggered cough. We investigated the role of the cAMP-elevating adenylate cyclase (CyaA) and pertussis (PT) toxins in the upregulation of mucin production in B. pertussis-infected airway epithelia. Using human pseudostratified airway epithelial cell layers cultured at air-liquid interface (ALI), we show that purified CyaA and PT toxins (100 ng/mL) can trigger production of the major airway mucins Muc5AC and Muc5B. Upregulation of mucin secretion involved activation of the cAMP response element binding protein (CREB) and was blocked by the 666-15-Calbiochem inhibitor of CREB-mediated gene transcription. Intriguingly, a B. pertussis mutant strain secreting only active PT and producing the enzymatically inactive CyaA-AC- toxoid failed to trigger any important mucus production in infected epithelial cell layers in vitro or in vivo in the tracheal epithelia of intranasally infected mice. In contrast, the PT- toxoid-producing B. pertussis mutant secreting the active CyaA toxin elicited a comparable mucin production as infection of epithelial cell layers or tracheal epithelia of infected mice by the wild-type B. pertussis secreting both PT and CyaA toxins. Hence, the cAMP-elevating activity of B. pertussis-secreted CyaA was alone sufficient for activation of mucin production through a CREB-dependent mechanism in B. pertussis-infected airway epithelia in vivo.

• MeSH
• adenylátcyklasový toxin toxicita   MeSH
• Bordetella pertussis metabolismus   patogenita   MeSH
• buněčné linie MeSH
• dýchací soustava metabolismus   mikrobiologie   MeSH
• epitelové buňky metabolismus   mikrobiologie   MeSH
• lidé MeSH
• mucin 5AC metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• pertuse metabolismus   mikrobiologie   MeSH
• protein vázající cAMP responzivní element metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• adenylátcyklasový toxin * MeSH
• adenylátcyklasy MeSH
• aktivace enzymů MeSH
• cholerový toxin MeSH
• faktory virulence rodu Bordetella * MeSH
• fosfolipasy MeSH
• pertusový toxin MeSH
• Publikační typ
• dopisy MeSH
• komentáře MeSH

Cíl práce: V diferenciální diagnostice dlouhodobého dráždivého kašle je třeba i u očkovaných pomýšlet na pertusi. Sérologické vyšetření, prováděné zejména v pozdní fázi onemocnění, nedává vždy jednoznačnou odpověď. Položili jsme si otázku, zda kvantitativní vysetření IgG protilátek k pertusovému toxinu (IgG-PT) napomůže při stanovení diagnózy. Materiál a metody: Od 1.1. 2005 do 30. 6. 2005 bylo sérologicky vyšetřeno celkem 139 dětí s dráždivým kašlem nebo dětí po aplikaci acelulární pertusové vakcíny. Porovnávala se sérologická odpověď aglutinačních protilátek a IgG-PT. Výsledky: Po očkováni tvořily děti protilátky v různé výši a ne vždy souhlasně v obou typech sérologické odpovědi. Děti s klinickými projevy pertuse tvořily protilátky dobře, avšak oba typy protilátek často v nezměněné výši dlouhodobě přetrvávaly. Závěr. Stanovení IgG-PT může diagnózu onemocnění podpořit, avšak samotné vyšetření jednoznačně pertusi potvrdit nedokáže.

Background: In the differential diagnosis of protracted irritating cough we should always consider the possibility of pertussis. Serology performed primarily in a late stage of the disorder does not always provide a clear answer. We wanted to verity whether a quantitative determination of IgG antibodies to the pertussis toxin (IgG-PT) could help establish a clear diagnosis. Material and methods: Between 1 January and 30 June 2005 we /e performed serological investigations in 139 children presenting with an irritable cough or after application of an acellular pertussis vaccine. In 95 children we compared the serological response of agglutination antibodies and IgG-PT. Results: After vaccination the children presented with different levels of antibodies and these were not always identical in two types of serological response. Children with clinical manifestations of pertussis presented a good formation of antibodies, but the two kinds of antibodies often persisted for long periods at unchanged levels. Conclusions: The determination of IgG-PT can assist the diagnosis of the disease, but this investigation alone cannot yield a clear-cut confirmation of pertussis.

• MeSH
• dítě MeSH
• lidé MeSH
• pertuse diagnóza   MeSH
• pertusový toxin imunologie   MeSH
• sérologické testy MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH

• MeSH
• adenylátcyklasový toxin MeSH
• antigeny CD11b imunologie   MeSH
• antigeny CD18 imunologie   MeSH
• Bordetella pertussis patogenita   MeSH
• faktory virulence rodu Bordetella MeSH
• integriny imunologie   MeSH
• pertusový toxin MeSH

The Bordetella adenylate cyclase-hemolysin (CyaA, ACT, or AC-Hly) is a multifunctional toxin. Simultaneously with promoting calcium ion entry, CyaA delivers into host cells an adenylate cyclase enzyme (AC) and permeabilizes cell membrane by forming small cation-selective pores. Indirect evidence suggested that these two activities were accomplished by different membrane-inserted CyaA conformers, one acting as an AC-delivering monomer and the other as an uncharacterized pore-forming oligomer. We tested this model by directly detecting toxin oligomers in cell membrane and by assessing oligomerization of specific mutants with altered pore-forming properties. CyaA oligomers were revealed in sheep erythrocyte membranes by immunogold labeling and directly demonstrated by pulldown of membrane-inserted CyaA together with biotinylated CyaA-AC(-) toxoid. Membrane oligomers of CyaA could also be resolved by nondenaturing electrophoresis of mild detergent extracts of erythrocytes. Furthermore, CyaA mutants exhibiting enhanced (E581K) or reduced (E570K+E581P) specific hemolytic and pore-forming activity were found to exhibit also a correspondingly enhanced or reduced propensity to form oligomers in erythrocyte membranes. On the other hand, processed CyaA, with the AC domain cleaved off by erythrocyte proteases, was detected only in a monomeric form excluded from the oligomers of unprocessed CyaA. These results provide the first direct evidence that oligomerization is involved in formation of CyaA pores in target membranes and that translocation of the AC domain across cell membrane may be accomplished by monomeric CyaA.

• MeSH
• adenylátcyklasový toxin farmakokinetika   metabolismus   MeSH
• Bordetella enzymologie   MeSH
• endocytóza MeSH
• erytrocyty MeSH
• hemolýza účinky léků   MeSH
• missense mutace MeSH
• multimerizace proteinu MeSH
• ovce MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Myeloid phagocytes have evolved to rapidly recognize invading pathogens and clear them through opsonophagocytic killing. The adenylate cyclase toxin (CyaA) of Bordetella pertussis and the edema toxin (ET) of Bacillus anthracis are both calmodulin-activated toxins with adenylyl cyclase activity that invade host cells and massively increase the cellular concentrations of a key second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP). However, the two toxins differ in the kinetics and mode of cell entry and generate different cAMP concentration gradients within the cell. While CyaA rapidly penetrates cells directly across their plasma membrane, the cellular entry of ET depends on receptor-mediated endocytosis and translocation of the enzymatic subunit across the endosomal membrane. We show that CyaA-generated membrane-proximal cAMP gradient strongly inhibits the activation and phosphorylation of Syk, Vav, and Pyk2, thus inhibiting opsonophagocytosis. By contrast, at similar overall cellular cAMP levels, the ET-generated perinuclear cAMP gradient poorly inhibits the activation and phosphorylation of these signaling proteins. Hence, differences in spatiotemporal distribution of cAMP produced by the two adenylyl cyclase toxins differentially affect the opsonophagocytic signaling in myeloid phagocytes.

• MeSH
• adenylátcyklasový toxin toxicita   MeSH
• AMP cyklický metabolismus   MeSH
• antigeny bakteriální toxicita   MeSH
• bakteriální toxiny toxicita   MeSH
• časoprostorová analýza MeSH
• fagocytóza účinky léků   MeSH
• fagocyty účinky léků   metabolismus   MeSH
• fosforylace účinky léků   MeSH
• lidé MeSH
• mikrofilamenta účinky léků   MeSH
• opsoniny farmakologie   MeSH
• receptory imunologické metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• THP-1 buňky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Monocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells. Bordetella pertussis, the pertussis agent, secretes an adenylate cyclase toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producing B. pertussis bacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA toxin provoked loss of macrophage marker expression on in vitro differentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiated in vitro The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating toxin action may represent a novel immune evasion strategy of bacterial pathogens.IMPORTANCE Macrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agent Bordetella pertussis The adenylate cyclase toxin (CyaA) mediates immune evasion of B. pertussis by suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.

• MeSH
• adenylátcyklasový toxin škodlivé účinky   metabolismus   MeSH
• alveolární makrofágy účinky léků   metabolismus   MeSH
• Bordetella pertussis chemie   MeSH
• buněčná diferenciace účinky léků   MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• monocyty účinky léků   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The interaction of Bordetella pertussis adenylate cyclase toxin (CyaA) with complement receptor 3 (CR3, CD11b/CD18) involves N-linked oligosaccharide chains. To investigate the relative importance of the individual N-glycans of CR3 for toxin activity, the asparagine residues of the consensus N-glycosylation sites of CR3 were substituted with glutamine residues that cannot be glycosylated. Examination of CR3 mutant variants and mass spectrometry analysis of the N-glycosylation pattern of CR3 revealed that N-glycans located in the C-terminal part of the CD11b subunit are involved in binding and cytotoxic activity of CyaA. We suggest that these N-glycans form a defined clustered saccharide patch that enables multivalent contact of CR3 with CyaA, enhancing both affinity and specificity of the integrin-toxin interaction.

• MeSH
• adenylátcyklasový toxin genetika   metabolismus   MeSH
• antigeny CD11b chemie   metabolismus   MeSH
• antigeny CD18 chemie   metabolismus   MeSH
• asparagin genetika   MeSH
• Bordetella pertussis metabolismus   patogenita   MeSH
• glutamin genetika   MeSH
• glykosylace MeSH
• lidé MeSH
• makrofágový antigen 1 genetika   metabolismus   MeSH
• polysacharidy metabolismus   MeSH
• substituce aminokyselin MeSH
• terciární struktura proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH